SummaryThe completion of the Arabidopsis genomic sequence offers the possibility to extract global information about regulatory mechanisms. Here, we describe a data mining strategy in combination with gene expression analysis to identify bona fide genes regulated by the E2F transcription factor. Starting with a genomewide search of chromosomal sites containing E2F-binding sites, we studied in depth two of the most abundant E2F-binding sites within the Arabidopsis genome and identified over 180 potential E2F target genes. Among them and in addition to cell cycle-related genes, we have also identified genes belonging to other functional categories, e.g. transcription, stress and defense or signaling. We have determined the expression levels of genes selected from different categories under two experimental situations. Using cultured cells partially synchronized with aphidicolin, we found that most potential E2F targets identified in silico show a cell cycle-regulated expression pattern with a peak in early/mid S-phase. In addition, we used Arabidopsis transgenic plants expressing a DP gene containing a truncated DNA-binding domain, which likely has a dominant-negative effect on AtE2Fa, b and c (also named AtE2F3, 1 and 2, respectively), which require DP for efficient DNA binding. Contrary to the up-regulation observed in early/mid S-phasecultured cells, the expression of a large number of potential E2F targets was decreased in the transgenic plants. Our results strongly support that the RBR/E2F pathway plays a crucial role in regulating the expression of the genes identified in this study.

Rupture of the seed coat and rupture of the endosperm are separate events in the germination of Nicotiana tabacum 1. cv Havana 425 seeds. Treatment with 1 O-5 M abscisic acid (ABA) did not appreciably affect seed-coat rupture but greatly delayed subsequent endosperm rupture by more than 100 h and resulted in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue. Therefore, ABA appears to act primarily by delaying endosperm rupture and radicle emergence. Measurements of P-1,3-glucanase activity, antigen content, and mRNA accumulation together with reporter gene experiments showed that induction of class I P-1,3-glucanase genes begins just prior to the onset of endosperm rupture but after the completion of seed-coat rupture. This induction was localized exclusively in the micropylar region of the endosperm, where the radicle will penetrate. ABA treatment markedly inhibited the rate of /3-1,3-glucanase accumulation but did not delay the onset of induction. lndependent of the ABA concentration used, onset of endosperm rupture was correlated with the same P-1,3-glucanase content/seed. These results suggest that ABA-sensitive class I p-1,3-glucanases promote radicle penetration of the endosperm, which is a key limiting step in tobacco seed germination.

The balance between cell proliferation and differentiation is crucial in multicellular organisms, where it is regulated by complex gene expression networks. This is particularly relevant in plants because organogenesis is a continuous postembryonic process. Here, we investigate the function of Arabidopsis thaliana E2Ff, an atypical member of the E2F family of transcription factors, which acts independently of a dimerization partner. We have focused our analysis on roots and hypocotyls, organs where (1) cell proliferation and differentiation are spatially and/or temporally separated, (2) growth depends on cell expansion in the longitudinal axis, and (3) the AtE2Ff promoter is active. AtE2Ff overexpression produced a reduction in the size of differentiated cells of these organs. Cells of mutant e2ff-1 plants with reduced levels of AtE2Ff mRNA were larger, especially in the hypocotyl, suggesting a role as a growth regulator. These effects of AtE2Ff are not associated with changes in nuclear ploidy levels or in the expression of cell cycle marker genes. However, expression of a subset of cell wall biogenesis genes is misregulated in an AtE2Ff-dependent manner, and based on chromatin immunoprecipitation experiments, they seem to be direct E2F targets. Our results highlight the complex regulatory function exerted by E2F and suggest a possible role of AtE2Ff in repressing cell wall biosynthesis genes during cell elongation in differentiated cells.