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 - 4.03

FUNCTIONAL ANALYSIS OF THE NF-YB6 GENE FROM ARABIDOPSIS

K. PETRONI, P. GALESI, G. GUSMAROLI, C. TONELLI

Università degli Studi di Milano, Dipartimento di Scienze Biomolecolari e Biotecnologie, Via Celoria 26, 20133 Milano

Transcription factors, CAAT binding proteins, NF-Y, Arabidopsis

In yeast and mammals, the CCAAT box is recognized by NF-Y, a trimer composed of distinct subunits: NF-YA, NF-YB and NF-YC, all required for DNA-binding and encoded by single copy genes (1). In vertebrates, NF-Y promotes and/or stabilises the DNA-binding of many classes of neighbouring transcription factors, sited on adjacent DNA elements, but it has been also proposed a role for NF-Y in chromatin remodelling and nucleosome displacement. Thus, the NF-Y complex could be a key factor in the regulation of the eukaryotic genome during development and differentiation.

A systematic screening of the Arabidopsis thaliana NF-Y homologues led to the identification and cloning of the complete NF-Y family in Arabidopsis, composed of 29 genes: 10 NF-YAs, 10 NF-YBs and 9 NF-YCs, sharing a degree of homology higher than 75% at aminoacidic level with the corresponding yeast and mammal genes (2,3). Their role is largely unknown, except for AtNF-YB9, which corresponds to the Leafy cotyledon1 gene (LEC1), required for embryo maturation and for specification of cotyledon identity in Arabidopsis (4).

Expression profiling of 29 NF-Y transcription factors from Arabidopsis performed by RT-PCR on RNA samples from several organs and developmental stages showed how, inside each family, certain members are expressed ubiquitously, during both vegetative and reproductive growth, while others are transcribed only in specific tissues or developmental stages, in addition to considerable differences in their relative expression levels. In agreement with previous data AtNF-YB9, corresponding to Leafy Cotyledon 1, is induced just in green siliques, during the early embryo developmental stages.

Based on phylogenetic analysis AtNF-YB6 resulted to be the NF-Y gene more similar to LEC1. Similarly to LEC1, NF-YB6 is expressed in siliques during embryo development and differs from the others at two lysine positions -K78D and K107Q- that are predicted to be important for DNA-binding functions.

Transgenic plants in which the AtNF-YB6 gene is silenced or ectopically expressed have been already produced. T2 overexpression lines show a reduced germination rate and an altered morphology of seedlings. In particular, the hypocotyl and the attachment region of cotyledons are elongated; cotyledons are not fully expanded and often asymmetrical. The first true leaves are characterized by asymmetry both in size and in orientation, while adult plants show distorted rosette leaves. Interestingly, T2 antisense plants show the same phenotype.

Recently, AtNF-YB6 has been indicated to have a specific role in embryo development, since 7% of RNAi lines for this gene have 30% of defective seeds with embryos blocked at different developmental stages, such as the globular one (5). We are currently analyzing five T-DNA insertion mutants of the AtNFY-B6 gene identified among available collections through database search. One of these carries a T-DNA positioned upstream of ATG, three alleles have a T-DNA insertion inside the coding region, while the fifth one is located downstream the ORF a few base pairs before the polyA signal.

(1) Maity and de Crombrugghe (1998), Trends Biochem. Sci. 23: 174-178.

(2) Gusmaroli et al., (2001) Gene 264: 173-185.

(3) Gusmaroli et al., (2002) Gene 283:41-48.

(4) Lotan et al. (1998), Cell 93:1195-1205.

(5) Kwong et al. (2003) Plant Cell 15:5-18.