We describe the Arabidopsis gene DAG2 encoding a Dof zinc finger protein and show that it is involved in the control of seed germination. An Arabidopsis mutant line with a T-DNA insertion in DAG2 isolated by reverse genetics produces seeds that are substantially more dependent than the wild type on the physical stimuli-light and cold treatment-that promote germination. Mutant dag2 seeds also are less sensitive to the germination-promotive effect of gibberellins, because a 10-fold higher amount of gibberellins is needed to restore germination when endogenous gibberellin biosynthesis is blocked. The seed germination characteristics of the dag2 mutant are opposite to those of dag1 , a knockout mutant of another Dof gene ( DAG1 ) that we showed previously to be involved in the control of seed germination, and are similar to those of plants that overexpress DAG1 . The promoter of the DAG2 gene is active specifically in the vascular system of the mother plant but not in the embryo, and segregation analysis indicates that the effect of the dag2 mutation is maternal. Both characteristics are in common with DAG1 ; additionally, the DAG1 and DAG2 proteins share high sequence homology and an identical zinc finger domain. These data suggest, and the germination phenotype of the double mutant is compatible with, a model whereby the zinc finger proteins DAG1 and DAG2 act on a maternal switch that controls seed germination, possibly by regulating the same gene(s).

The Dof proteins are a large family of plant transcription factors that share a single highly conserved zinc finger. The tobacco Dof protein NtBBF1 was identified by its ability to bind to regulatory domain B in the promoter of the rolB oncogene. In this study, we show that the ACT TTA target sequence of NtBBF1 in domain B is necessary for tissue-specific expression of rolB . ␤ -Glucuronidase (GUS) activity of tobacco plants containing a rolB promoter-GUS fusion with a mutated NtBBF1 target sequence within domain B is almost completely suppressed in apical meristems and is severely abated in the vascular system. The ACTTTA motif is shown here also to be one of the cis -regulatory elements involved in auxin induction of rolB . The pattern of NtBBF1 expression in plants is remarkably similar to that of rolB , except in mesophyll cells of mature leaves, in which only NtBBF1 expression could be detected. Ectopic expression of rolB in mesophyll cells was achieved by particle gun delivery if the NtBBF1 binding sequence was intact. These data provide evidence that in the plant, a Dof protein DNA binding sequence acts as a transcriptional regulatory motif, and they point to NtBBF1 as the protein involved in mediating tissue-specific and auxin-inducible expression of rolB . INTRODUCTIONInfection of dicotyledonous plants with Agrobacterium rhizogenes results in hairy root disease (Elliot, 1951). Hairy root causes an abundant proliferation of neoplastic roots containing T-DNA from the root-inducing (Ri) bacterial plasmid (Chilton et al., 1982;Spanò et al., 1982;White et al., 1982;Willmitzer et al., 1982). In the T-DNA sequence, 18 open reading frames have been identified (Slightom et al., 1986). Four of these, termed the rol oncogenes, coincide with genetic loci previously localized by transposon mutagenesis (White et al., 1985). Among the rol oncogenes, rolB is the only one that, when inactivated, totally suppresses root induction by A. rhizogenes (White et al., 1985). In addition, rolB is the only individual gene capable of inducing growth of transformed roots for all plants tested (Cardarelli et al., 1987;Spena et al., 1987;Capone et al., 1989). More recently, in cultured thin cell layers ( Tran Thanh Van et al., 1974), the stimulation of meristem formation has been shown to be the primary effect of rolB (Altamura et al., 1994). Expression of rolB greatly increases the sensitivity of transformed plant cells to auxin (Spanò et al., 1988;Maurel et al., 1991), and tyrosine phosphatase activity has been associated with the RolB protein when it is expressed in Escherichia coli (Filippini et al., 1996), suggesting that RolB may act by perturbing the auxin signal transduction pathway.The expression of rolB in transgenic plants also has been extensively analyzed and found to be strongest in root and shoot meristems (Schmülling et al., 1989;Maurel et al., 1990;Altamura et al., 1991;Capone et al., 1991), which is in accord with an effect of the oncogene on meristem induction. Expression was also detectable in the vascular system....

Recombinant DNA libraries were constructed for seven chromosome types isolated from two translocation lines of field bean (Vicia faba L.) with reconstructed karyotypes. The chromosomes were selected so that the set of libraries covers the whole V. faba genome more than once. Individual chromosome types were highly purified by flow sorting, and their DNA was amplified by degenerate oligonucleotide-primed (DOP) polymerase chain reaction (PCR) and cloned into a plasmid vector. The choice of restriction site present in PCR primer and refinement of cloning protocol resulted in high cloning efficiency and allowed generation of libraries consisting of about 10(5) clones from 250 or 1000 sorted chromosomes. The insert size ranged between 50 and 2200 bp and the mean length estimated in individual libraries varied between 310 and 487 bp. Hybridization of cloned fragments with labelled genomic DNA showed that about 60% of inserts represented unique or low-copy sequences. The suitability of the libraries for genome mapping was demonstrated by isolation of clones containing microsatellite motifs.

A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1-10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G1/S interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 μM). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of 1×10(5)/root and 1.4×10(5)/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.

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