Previously we have shown that overexpression of the heterodimeric E2Fa-DPa transcription factor in Arabidopsis thaliana results in ectopic cell division, increased endoreduplication, and an early arrest in development. To gain a better insight into the phenotypic behavior of E2Fa-DPa transgenic plants and to identify E2Fa-DPa target genes, a transcriptomic microarray analysis was performed. Out of 4,390 unique genes, a total of 188 had a twofold or more up- (84) or down-regulated (104) expression level in E2Fa-DPa transgenic plants compared to wild-type lines. Detailed promoter analysis allowed the identification of novel E2Fa-DPa target genes, mainly involved in DNA replication. Secondarily induced genes encoded proteins involved in cell wall biosynthesis, transcription and signal transduction or had an unknown function. A large number of metabolic genes were modified as well, among which, surprisingly, many genes were involved in nitrate assimilation. Our data suggest that the growth arrest observed upon E2Fa-DPa overexpression results at least partly from a nitrogen drain to the nucleotide synthesis pathway, causing decreased synthesis of other nitrogen compounds, such as amino acids and storage proteins.

Introduction

Progression through the cell cycle is essential for the continued existence of all uni- and multicellular organisms. It is crucial for the survival of a cell that its DNA is correctly replicated. In mammals, the onset of DNA replication is regulated by the activity of the heterodimeric E2F-DP transcription factor. The mammalian E2F family contains six proteins (E2F1, E2F2, E2F3, E2F4, E2F5 and E2F6) (Trimarchi and Lees, 2002). All E2Fs have an N-terminally located DNA-binding domain immediately followed by a dimerization domain, allowing them to pair with a dimerization partner (DP1 or DP2). Dimerization of E2F with DP is a prerequisite for high affinity, sequence-specific binding to the E2F consensus DNA-binding site. E2F activity is negatively regulated by retinoblastoma (Rb), which binds to the transcriptional activation domain of the E2F-DP factor, rendering it inactive. Moreover, the recruitment by Rb of DNA-modifying enzymes, such as histone deacetylases and polycomb proteins, leads to chromatin condensation with suppression of promoter activity of E2F-DP target genes as a result. Phosphorylation of Rb by cyclin-dependent kinases (CDKs) counteracts its inhibitory function, resulting in the release of transcriptionally active E2F-DP and consequential onset of DNA replication.

The mechanism of DNA replication seems to be conserved between mammals and plants, because E2F and DP genes have been isolated from different plant species, including wheat, tobacco, carrot, Arabidopsis and rice (Ramírez-Parra et al., 1999; Sekine et al., 1999; Albani et al., 2000; Magyar et al., 2000; Ramirez-Parra and Gutierrez, 2000; Kosugi and Ohashi, 2002a). In the Arabidopsis genome there are three E2F (E2Fa, E2Fb and E2Fc) and two DP (DPa and DPb) genes (Vandepoele et al., 2002). Recently, we have analyzed the phenotypes of plants co-overexpressing the E2Fa-DPa genes (De Veylder et al., 2002). Transgenic plants were smaller than control plants, had curled leaves and cotyledons, and were arrested in growth at an early stage of development. Microscopic analysis revealed that E2Fa-DPa-overproducing cells underwent ectopic cell division or endoreduplication, depending on the cell type. Whereas extra cell divisions resulted in cells smaller than those seen in the same tissues of control plants, supplementary endoreduplication caused the formation of giant nuclei. By using reverse transcription (RT)PCR, we demonstrated that the expression levels of genes involved in DNA replication (CDC6, ORC1, MCM and DNA pol α) were strongly up-regulated (De Veylder et al., 2002).

Physiologically important targets of the mammalian E2F-DP transcription factors have been identified by microarray hybridization experiments, chromatin immunoprecipitations and computer-assisted prediction (Ishida et al., 2001; Kel et al., 2001; Müller et al., 2001; Weinmann et al., 2001; Ren et al., 2002). E2F-DP-responsive genes can be found among genes involved in cell division, DNA repair and replication, mitotic progression, apoptosis and differentiation. Although little is known about the plant E2F-DP target genes, a database search has been published recently, in which the Arabidopsis genome was screened for genes harboring the TTTCCCGCC cis-acting element in their promoter (Ramirez-Parra et al., 2003). However, it is still unclear whether this specific cis-acting element is the only one recognized by the plant E2F-DP complexes, or whether the presence of the TTTCCCGCC element is sufficient to mark a gene as a true E2F-DP target gene. In order to identify the functional classes of genes regulated by E2Fa-DPa and to understand the nature of the phenotype of the E2Fa-DPa-overexpressing plants, we designed a microarray experiment that compared the transcript levels of 4,571 genes of wild-type and transgenic lines. We found distinct classes of genes that were up- or down-regulated in the E2Fa-DPa plants. Promoter analysis allowed us to distinguish among the downstream expressed genes, the genes that were putatively under direct control of E2Fa-DPa. Furthermore, we found that the increased expression levels of E2Fa-DPa have a large impact on the expression levels of genes involved in nitrogen assimilation and metabolism.

Materials and Methods

Plant material

Double transgenic CaMV35S-E2Fa-DPa plants were obtained by crossing homozygous CaMV35S-E2Fa and CaMV35S-DPa plants (De Veylder et al., 2002). Double transformants were grown under a 16- hour light/8-hour dark photoperiod at 22°C on germination medium (Valvekens et al., 1988).

Construction of microarrays

The Arabidopsis thaliana (L.) Heynh. microarray consisted of 4,608 cDNA fragments spotted in duplicate, distant from each other, on Type V silane-coated slides (Amersham Biosciences, Little Chalfont, UK). The clone set included 4,571 Arabidopsis cDNAs from the unigene clone collection Arabidopsis Gem I (Incyte Genomics, Palo Alto, CA). The functional annotation of the genes related to the spotted cDNAs was retrieved by BLASTN against genomic sequences. To facilitate the analysis, a collection of genomic sequences was built each bearing only one gene. In each of these sequences, the upstream intergenic sequence was followed by the exon-intron structure of the gene and the downstream intergenic sequence, or, in other words, the whole genomic sequence between start and stop codons from neighboring protein-encoding genes. From the BLASTN output, the best hits were extracted and submitted to a BLASTX search against protein databases. From this analysis, the set of 4,571 cDNAs appeared to constitute 4,390 unique clones. To obtain more detailed information concerning the potential function of the genes, protein domains were searched using ProDom. The complete set can be found at http://www.psb.ugent.be/E2F/. The cDNA inserts were amplified by PCR with M13 primers, purified with MultiScreen- PCR plate (Millipore, Bedford, MA), and arrayed on slides using a Generation III printer (Amersham Biosciences). Slides were blocked in 3.5% SSC (1×SSC, 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 0.2% sodium dodecyl sulfate (SDS), 1% bovine serum albumin for 10 minutes at 60°C.

RNA amplification and labeling

Antisense RNA was amplified with a modified protocol of in vitro transcription (Puskás et al., 2002). For the first-strand cDNA synthesis, 5 μg of total RNA was mixed with 2 μg of a HPLC-purified anchored oligo(dT) + T7 promoter (5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24(ACG)-3′) (Eurogentec, Seraing, Belgium), 40 units of RNAseOUT (Invitrogen, Gaithersburg, MD) and 0.9 M D(+)trehalose (Sigma-Aldrich, St. Louis, MO) in a total volume of 11 μl and heated to 75°C for 5 minutes. To this mixture, 4 μl 5×first-strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol, 1 μl 10 mM dNTP mix, 1 μl 1.7 M D(+)trehalose (Sigma-Aldrich), and 1 μl SuperScript II (Invitrogen) were added to 20 μl final volume. The sample was incubated in a UnoII thermocycler (Whatman Biometra, Göttingen, Germany) at 37°C for 5 minutes, at 45°C for 10 minutes, 10 cycles at 60°C for 2 minutes and at 55°C for 2 minutes. To the first-strand reaction mix, 103.8 μl water, 33.4 μl 5×second-strand synthesis buffer (Invitrogen), 3.4 μl 10 mM dNTP mix, 1 μl of 10 U/μl DNA ligase (Invitrogen), 4 μl 10 U/μl DNA Polymerase I (Invitrogen), and 1 μl 2 U/μl RNAse H (Invitrogen) were added and incubated at 16°C for 2 hours. The synthesized double-stranded cDNA was purified with Qiaquick (Qiagen, Hilden, Germany). Antisense RNA was synthesized by AmpliScribe T7 high-yield transcription kit (Epicentre Technologies, Madison, WI) in a total volume of 20 μl according to the manufacturer's instructions. The RNA was purified with the RNeasy purification kit (Qiagen). From this RNA, 5 μg was labeled by reverse transcription using random nonamer primers (Genset, Paris, France), 0.1 mM d(G/T/A)TPs, 0.05 mM dCTP (Amersham Biosciences), 0.05 mM Cy3-dCTP or Cy5-dCTP (Amersham Biosciences), 1×first-strand buffer, 10 mM dithiothreitol, and 200 U of SuperScript II (Invitrogen) in 20 μl total volume. The RNA and primers were denatured at 75°C for 5 minutes and cooled on ice before the remaining reaction components were added. After 2 hours incubation at 42°C, mRNA was hydrolyzed in 250 mM NaOH for 15 minutes at 37°C. The sample was neutralized with 10 μl of 2 M 3-(N-morpholino)propanesulfonic acid and purified with Qiaquick (Qiagen).

Array hybridization and post-hybridization processes

The probes were resuspended in 30 μl hybridization solution (50% formamide, 5×SSC, 0.1% SDS, 100 μg/ml salmon sperm DNA) and prehybridized with 1 μl poly(dT) (1 mg/ml) at 42°C for 30 minutes to block hybridization on the polyA/T tails of the cDNA on the arrays. Mouse COT DNA (1 mg/ml) (Invitrogen) was added to the mixture and placed on the array under a glass coverslip. Slides were incubated for 18 hours at 42°C in a humid hybridization cabinet (Amersham Biosciences). Post-hybridization washing was performed for 10 minutes at 56°C in 1×SSC, 0.1% SDS, twice for 10 minutes at 56°C in 0.1×SSC, 0.1% SDS, and for 2 minutes at 37°C in 0.1×SSC.

Scanning and data analysis

Arrays were scanned at 532 nm and 635 nm using a Generation III scanner (Amersham Biosciences). Image analysis was performed with ArrayVision (Imaging Research Inc, St. Catharines, Ontario, Canada). Spot intensities were measured as artifact-removed total intensities (ARVol) without correction for background. We first addressed withinslide normalization by plotting for each single slide a `MA-plot' (Yang et al., 2002), where M=log2 (R/G) and

\(\mathrm{A}=\mathrm{log}_{2}\sqrt{\mathrm{R}{\times}\mathrm{G}}\)
⁠. Dye intensity differences were corrected with the `LOWESS' normalization. Subsequently, between-slide normalization and differentially expressed gene identification between the two genotypes were performed by sequential analysis of variances (ANOVAs), according to the method of Wolfinger et al. (Wolfinger et al., 2001). (i) The base- 2 logarithm of the `LOWESS'-transformed measurements for all 73,136 spots (yiklm) were subjected to a normalization model yiklm=μ+Ak +AkDlRmiklm, where μ is the sample mean, Ak the effect of the kth array (k=1-4), AkDlRm the channel effect (AD) for the mth replication (m=2; left and right) of the total collection of i (i=1,..., 4571) cDNA fragments, and ϵiklm the stochastic error. (ii) The residuals from this model were subjected to 4,571 gene-specific models rijkl=μ+GiAk+GiDl+GiCjijkl, where GiAk is the spot effect, GiDl the gene-specific dye effect, GiCj the signal intensity for genes that can specifically be attributed to the genotypes (effect of interest), and γijkl the stochastic error. All effects were assumed to be fixed, except for ϵiklm and γijkl. T-tests for differences between the GiCj effects were performed, all based on n1+n2-6 degrees of freedom, where n1 and n2 correspond to the number of wild-type and E2Fa-DPa hybridizations, respectively. Bonferroni adjustment for the 4,571 tests to assure an experiment-wise false positive rate of 0.05 results in a P-value cut-off of 1e-5.0, which is certainly too conservative. Thus, no further adjustments for multiple testing were done. Therefore, we chose to set the P-value cut-off arbitrarily at the 0.05 level. We used Genstat for both the normalization and gene model fits.

RT-mediated PCR analysis

RNA was isolated from plants 8 days after sowing with the Trizol reagent (Amersham Biosciences). First-strand cDNA was prepared from 3 μg of total RNA with the Superscript RT II kit (Invitrogen) and oligo(dT)18 according to the manufacturer's instructions. A 0.25 μl aliquot of the total RT reaction volume (20 μl) was used as a template in a semi-quantitative RT-mediated PCR amplification, ensuring that the amount of amplified product remained in linear proportion to the initial template present in the reaction. From the PCR reaction, 10 μl was separated on a 0.8% agarose gel and transferred onto Hybond N+ membranes (Amersham Biosciences) that were hybridized at 65°C with fluorescein-labeled probes (Gene Images random prime module; Amersham Biosciences). The hybridized bands were detected with the CDP Star detection module (Amersham Biosciences). Primers used were 5′-AAAAAGCAGGCTGTGTCGTACGATCTTCTCCCGG-3′ and 5′-AGAAAGCTGGGTCATGTGATAGGAGAACCAGCG-3′ for E2Fa, 5′-ATAGAATTCGCTTACATTTTGAAACTGATG-3′ and 5′-ATAGTCGACTCAGCGAGTATCAATGGATCC-3′ for DPa, 5′-CAGATCTTGTTAACCTTGACATCTCAG-3′ and 5′-GGGTCAAAAGATACAACCACACCAG-3′ for glutamine synthetase (GS), 5′-GGTTTACGAGCTACATGGCCC-3′ and 5′-GAGCAATCCGTTCAGCCTCC-3′ for glutamate synthase (GOGAT), 5′-GCGTTTGACCACTCTTGGAGAC-3′ and 5′-GAACGCCATTGAGAAAGTCCGC-3′ for histone acetylase HAT B, 5′-GTTACCGGCTCGACTTGAAGATC-3′ and 5′-GAATCGGAGGGAAAGTCTGACG-3′ for LOB domain protein 41, 5′-GTGTGGTTTCCAAGCTTTCCTACG-3′ and 5′-GGTGAAGGGACTAGCCTTGTGG-3′ for isocitrate lyase, 5′-GGGATCAATCCTCAGGAGAAGG-3′ and 5′-CCGTCCATCTTTATTAGCGGCATG-3′ for nitrite reductase (NiR), and 5′-TTACCGAGGCTCCTCTTAACCC-3′ and 5′-ACCACCGATCCAGACACTGTAC-3′ for actin 2 (ACT2).

Promoter analysis

The intergenic sequence corresponding to the promoter area of each gene spotted on the microarray was deduced from genomic sequences. From these intergenic sequences, up to 500 bp upstream of the ATG start codon were extracted and subjected to motif searches to retrieve potential E2F elements. Of the 4,571 expressed sequence tags (ESTs) spotted on the microarray, we could retrieve the genomic sequence of 4,390. This difference is due to the presence of duplicate genes and mitochondrial or chloroplast DNA on the microarray. Both the position and frequency of occurrence were determined with the publicly available MatInspector (version 2.2) by using matrices extracted from PlantCARE and matrices made especially for this particular analysis (Lescot et al., 2002). The relevance of each motif was evaluated against a background consisting of all the sequences from the dataset by using the Fisher exact test.

Results and Discussion

Experimental setup and statistical analysis

A microarray containing in replicate 4,571 Arabidopsis expressed sequence tags (ESTs) was used to compare the transcriptome of the wild-type with that of E2Fa-DPa-overexpressing plants. cDNA was synthesized from total RNA of plants harvested 8 days after sowing. At that stage, transgenic plants can be distinguished from control plants by the appearance of curled cotyledons that display ectopic cell divisions and enhanced endoreduplication (De Veylder et al., 2002). In the first two hybridizations, including a biological repeat, fluorescently Cy3- and Cy5-labeled probe pairs of control and E2Fa-DPa cDNAs were used. Subsequently, a dyeswap replication was performed for both hybridizations, resulting in a total of four cDNA microarray hybridizations. Because each cDNA was printed in duplicate on the array, eight data points for every gene were obtained.

Fluorescence levels were analyzed to establish whether the expression level of each gene varied according to the overexpression of the E2Fa-DPa transcription factor. Two sequential ANOVA models were used, as proposed by Wolfinger et al. (Wolfinger et al., 2001). First, the model called `normalization model' accounts for experiment-wise systematic effects, such as array and channel effects, which could bias inferences made on the data from the individual genes. The residuals from this model represent normalized values and are the input data for the second model, called the `gene' model. The gene models are fitted separately to the normalized data from each gene (see Materials and Methods). In this procedure, normalized expression levels rather than ratios are used as units.

For each of the 4,571 genes on the arrays the genotype-specific signal intensity was determined and t-tested for significant differences (P<0.05). Fig. 1 presents the P values obtained (as the negative log10 of the P value) against the magnitude of the effect (log2 of estimated fold change). This so-called volcano plot illustrates the substantial difference of significance testing as opposed to cut-offs strictly based on the fold change. The two vertical reference lines indicate a twofold cut-off for either repression or induction, whereas the horizontal reference line refers to the P-value cut-off at 0.05. These reference lines divide the plot into six meaningful sectors. The 3,126 genes in the lower middle sector have low significance and low fold change, and both methods are in agreement that the corresponding changes are not significant. The 188 genes in the upper left and right sectors have high significance (P<0.05) and high fold change (≥2); 84 of these genes show a significant two-or-more-fold induction of expression, whereas the remaining 104 genes show a significant two-or-more-fold repression of expression in the E2Fa-DPa plants. The identity of these genes was confirmed by sequencing, and the induction of a random set of selected genes was confirmed by RT-PCR analysis (Fig. 2). Finally, the 1,257 genes in the upper middle sector represent significant (P<0.05) up- or down-regulated genes, but with a low (≤2) fold change. The full dataset of genes can be viewed at http://www.psb.ugent.be/E2F/.

Fig. 1.

Volcano plot of significance against effect. Each x represents one of the 4,571 genes, with the negative log10 of the P value from the gene model plotted against the difference between least-square means for the genotype effect. The horizontal line represents the test-wise threshold of P=0.05. The two vertical reference lines indicate a twofold cut-off for either repression or induction.

Fig. 1.

Volcano plot of significance against effect. Each x represents one of the 4,571 genes, with the negative log10 of the P value from the gene model plotted against the difference between least-square means for the genotype effect. The horizontal line represents the test-wise threshold of P=0.05. The two vertical reference lines indicate a twofold cut-off for either repression or induction.

Fig. 2.

Verification of microarray analysis by RT-PCR. RT-PCR analysis was carried out under linear amplification conditions. The actin 2 gene (ACT2) was used as loading control. GS, glutamine synthetase; GOGAT, glutamate synthase; NiR, nitrite reductase.

Fig. 2.

Verification of microarray analysis by RT-PCR. RT-PCR analysis was carried out under linear amplification conditions. The actin 2 gene (ACT2) was used as loading control. GS, glutamine synthetase; GOGAT, glutamate synthase; NiR, nitrite reductase.

DNA replication and cell cycle genes

Genes up- or down-regulated in the E2Fa-DPa transgenic plants can be classified into clear groups according to their function (Tables 1 and 2). Among the genes that are twofold or more up-regulated, 14 belong to the class of DNA replication and modification, correlating with the observation that E2Fa-DPa-overexpressing plants undergo extensive endoreduplication. Most of these genes have previously been shown to be up-regulated by E2F-DP overexpression in mammalian cells, including a putative thymidine kinase, replication factor c, adenosylhomocysteinase, DNA (cytosine-5)-methyltransferase, and histone genes (Ishida et al., 2001; Müller et al., 2001; Ren et al., 2002). Other E2Fa-DPa-induced S phase genes include a linker histone protein, the topoisomerase 6 subunit A, and two subunits of the histone acetyltransferase HAT B complex, namely HAT B and Msi3. The HAT B complex is responsible for the specific diacetylation of newly synthesized histone H4 during nucleosome assembly on newly synthesized DNA (Lusser et al., 1999).

Table 1.

Arabidopsis genes up-regulated twofold or more in E2Fa-DPa plants sorted according to functional category

Gene identification Accession number ORF name Fold induction E2F motif Position* Strand
DNA replication and modification (14)        
Putative thymidine kinase   AI997851   At3g07800   8.44     
DNA methyltransferase   AI994691   At5g49160   5.37     
Msi3   AW004204   At4g35050   4.89   TTTCCCGC   −75   −  
Putative linker histone protein   AI994590   At3g18035   3.31     
Putative replication factor c   AI997934   At1g21690   3.30   TTTCCCGC   −96   −  
Topoisomerase 6 subunit A   AI995290   At5g02820   2.62   TTTCCCGC   −66   +  
     TTTGGCGG   −369   +  
Histone H4-like protein   AI999171   At3g46320   2.55   TTTGGCGC   −310   +  
Histone acetylase HAT B   AI998229   At5g56740   2.36   TTTCCCGC   −50   +  
Putative histone H1   AI996137   At1g06760   2.27     
Histone H2A-like protein   AI995882   At4g27230   2.23     
Putative DNA gyrase subunit A   AI995400   At3g10690   2.20   ATTCGCGC   −91   +  
     TTTGGCGG   −107   −  
Histone H2B-like protein   AI999101   At5g59910   2.16   ATTCCCGC   −329   −  
     ATTGGCGC   −303   −  
Putative mismatch binding protein   AI993280   At3g24320   2.10     
Adenosyl homocysteinase   AI996953   At4g13940   2.07     
Cell cycle (2)        
E2Fa   AJ294534   At2g36010   94.88     
CDKB1 ; 1   D10851   At3g54180   2.60   TTTCCCGC   −151   −  
Cell wall biogenesis (11)        
Xyloglucan endo-1,4-β-d-glucanase (meri-5)   AI994459   At4g30270   3.74     
Putative glycosyl transferase   AI999244   At1g70090   3.38     
α-Galactosyltransferase-like protein   AI998223   At3g62720   3.26     
Putative xyloglucan endotransglycosylase   AI999683   At3g23730   2.85     
Xyloglucan endo-1,4-β-d-glucanase-like protein   AI998301   At4g30280   2.74     
Putative xyloglucan endotransglycosylase   AI994477   At1g14720   2.51     
Putative glycosyl transferase   AI999770   At1g24170   2.39     
Putative UDP-glucose glucosyltransferase   AI997288   At1g22400   2.34   TTTCGCGC   −20   +  
Putative glucosyltransferase   AI998872   At2g15480   2.15     
Peroxidase   AI994622   At2g38380   2.11   TTTCGCGC   −314   −  
β-1,3-glucanase-like protein   AI994681   At3g55430   2.05     
Chloroplastic genes (7)        
Large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase   N96785   rbcL   4.71     
Ribosomal protein L33   AI994194   rp133   3.54   TTTCCCCC   −315   −  
Photosystem II protein   AW004203   PsbI   2.81     
Ribosomal protein L2   AW004266   rp12   2.61     
ATP-dependent protease subunit   AI997947   clpP   2.60     
Cytochrome B6   AI997102   PetB   2.55   TTTCGCGG   −36   −  
ATPase ϵ subunit   AW004251   atpE   2.17   TTTCCCGG   −160   −  
Mitochondrial genes (1)        
26S ribosomal RNA protein   AW004275   orf107a   2.87     
Transcription factors (6)        
LOB domain protein 41   AI996685   At3g02550   4.01     
WRKY transcription factor 21   AI992739   At2g30590   2.78   TTTCCCCC   −23   −  
GATA Zn-finger protein   AI995731   At3g16870   2.75     
Anthocyaninless2   AI993655   At4g00730   2.73     
Leucine zipper-containing protein   AI995691   At1g07000   2.43   TTTCCCCG   −33   +  
Homeodomain transcription factor (Athb-6)   AI999190   At2g22430   2.30     
Metabolism and biogenesis (11)        
Alcohol dehydrogenase   AI998773   At1g77120   5.09     
Putative isocitrate lyase   AI999168   At3g21720   3.08     
Protochlorophyllide reductase precursor   AI993342   At4g27440   2.39     
Sugar transporter-like protein   AI997793   At4g36670   2.27     
NADH-dependent glutamate synthase (GOGAT)   AI997600   At5g53460   2.25   TTTCGCCG   −225   +  
Nitrate reductase (NIA2)   AI996208   At1g37130   2.15     
Pectate lyase-like protein   AJ508995   At3g54920   2.13     
Putative sterol dehydrogenase   AI996340   At2g43420   2.10     
Glutamine synthetase root isozyme 1 (GS)   161G19T7   At1g66200   2.06     
Monosaccharide transporter STP3   AI997045   At5g61520   2.05     
Signal transduction (6)        
Calcium-dependent protein kinase   AI996555   At5g66210   2.96     
WD-40 repeat protein   AI993055   At5g14530   2.70   TTTCGCGG   −104   −  
Receptor-protein kinase-like protein   AI994727   At5g54380   2.59     
Putative phytochrome A   AI998146   At1g09570   2.45     
Putative leucine-rich receptor-like protein kinase   AI999651   At1g72180   2.13     
Putative receptor-like kinase   AI993298   At3g23750   2.06     
Others (13)        
Putative pollen allergen   AI996548   At3g45970   3.22     
Cold-regulated protein COR6,6   AW004198   At5g15970   3.03     
Phi-1-like protein   AI994601   At5g64260   2.60     
Lipid-transfer protein-like   AI998609   At5g01870   2.33     
DnaJ homologue   AI994551   At5g06910   2.32   ATTGGCGC   −103   +  
Blue copper-binding protein   AI996535   At5g20230   2.30     
Src-2-like protein   AI998679   Atlg09070   2.19     
Ring finger protein   AI999491   At3g61460   2.14     
Putative Tice22   AI993361   At3g23710   2.14     
Nodulin-like protein   AI996322   Atlg80530   2.07     
Putative resistance protein   AI997549   Atlg61100   2.06     
Seed imbitition protein-like   AI993446   At5g20250   2.05     
Putative disease resistance protein   AI998978   Atlg72900   2.04   TTTGGCGG   −175   +  
Unknown function (14)        
Putative protein   AI994686   At3g45730   5.14     
Putative protein   AI994734   At5g66580   3.18     
Unknown protein   AI999397   At2g38310   2.79   TTTGCCCC   −280   −  
Hypothetical protein   AI998042   Atlg57680   2.66     
Unknown protein   AI995465   At2g47440   2.50     
Unknown protein   AI994871   Atlg76970   2.34     
Hypothetical protein   AI998366   Atlg27500   2.21     
Putative protein   AI996967   At4g33050   2.20     
Putative protein   AI995917   At3g43690   2.18     
Unknown protein   AI993084   At2g25970   2.15     
Unknown protein   AI993077   Atlg68580   2.13     
Putative protein   AI993019   At5g14420   2.05   TTTCGCCG   −443   −  
Hypothetical protein   AI997428   Atlg57990   2.02     
Unknown protein   AI997827   At5g53740   2.01   TTTGGCGG   −66   +  
Gene identification Accession number ORF name Fold induction E2F motif Position* Strand
DNA replication and modification (14)        
Putative thymidine kinase   AI997851   At3g07800   8.44     
DNA methyltransferase   AI994691   At5g49160   5.37     
Msi3   AW004204   At4g35050   4.89   TTTCCCGC   −75   −  
Putative linker histone protein   AI994590   At3g18035   3.31     
Putative replication factor c   AI997934   At1g21690   3.30   TTTCCCGC   −96   −  
Topoisomerase 6 subunit A   AI995290   At5g02820   2.62   TTTCCCGC   −66   +  
     TTTGGCGG   −369   +  
Histone H4-like protein   AI999171   At3g46320   2.55   TTTGGCGC   −310   +  
Histone acetylase HAT B   AI998229   At5g56740   2.36   TTTCCCGC   −50   +  
Putative histone H1   AI996137   At1g06760   2.27     
Histone H2A-like protein   AI995882   At4g27230   2.23     
Putative DNA gyrase subunit A   AI995400   At3g10690   2.20   ATTCGCGC   −91   +  
     TTTGGCGG   −107   −  
Histone H2B-like protein   AI999101   At5g59910   2.16   ATTCCCGC   −329   −  
     ATTGGCGC   −303   −  
Putative mismatch binding protein   AI993280   At3g24320   2.10     
Adenosyl homocysteinase   AI996953   At4g13940   2.07     
Cell cycle (2)        
E2Fa   AJ294534   At2g36010   94.88     
CDKB1 ; 1   D10851   At3g54180   2.60   TTTCCCGC   −151   −  
Cell wall biogenesis (11)        
Xyloglucan endo-1,4-β-d-glucanase (meri-5)   AI994459   At4g30270   3.74     
Putative glycosyl transferase   AI999244   At1g70090   3.38     
α-Galactosyltransferase-like protein   AI998223   At3g62720   3.26     
Putative xyloglucan endotransglycosylase   AI999683   At3g23730   2.85     
Xyloglucan endo-1,4-β-d-glucanase-like protein   AI998301   At4g30280   2.74     
Putative xyloglucan endotransglycosylase   AI994477   At1g14720   2.51     
Putative glycosyl transferase   AI999770   At1g24170   2.39     
Putative UDP-glucose glucosyltransferase   AI997288   At1g22400   2.34   TTTCGCGC   −20   +  
Putative glucosyltransferase   AI998872   At2g15480   2.15     
Peroxidase   AI994622   At2g38380   2.11   TTTCGCGC   −314   −  
β-1,3-glucanase-like protein   AI994681   At3g55430   2.05     
Chloroplastic genes (7)        
Large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase   N96785   rbcL   4.71     
Ribosomal protein L33   AI994194   rp133   3.54   TTTCCCCC   −315   −  
Photosystem II protein   AW004203   PsbI   2.81     
Ribosomal protein L2   AW004266   rp12   2.61     
ATP-dependent protease subunit   AI997947   clpP   2.60     
Cytochrome B6   AI997102   PetB   2.55   TTTCGCGG   −36   −  
ATPase ϵ subunit   AW004251   atpE   2.17   TTTCCCGG   −160   −  
Mitochondrial genes (1)        
26S ribosomal RNA protein   AW004275   orf107a   2.87     
Transcription factors (6)        
LOB domain protein 41   AI996685   At3g02550   4.01     
WRKY transcription factor 21   AI992739   At2g30590   2.78   TTTCCCCC   −23   −  
GATA Zn-finger protein   AI995731   At3g16870   2.75     
Anthocyaninless2   AI993655   At4g00730   2.73     
Leucine zipper-containing protein   AI995691   At1g07000   2.43   TTTCCCCG   −33   +  
Homeodomain transcription factor (Athb-6)   AI999190   At2g22430   2.30     
Metabolism and biogenesis (11)        
Alcohol dehydrogenase   AI998773   At1g77120   5.09     
Putative isocitrate lyase   AI999168   At3g21720   3.08     
Protochlorophyllide reductase precursor   AI993342   At4g27440   2.39     
Sugar transporter-like protein   AI997793   At4g36670   2.27     
NADH-dependent glutamate synthase (GOGAT)   AI997600   At5g53460   2.25   TTTCGCCG   −225   +  
Nitrate reductase (NIA2)   AI996208   At1g37130   2.15     
Pectate lyase-like protein   AJ508995   At3g54920   2.13     
Putative sterol dehydrogenase   AI996340   At2g43420   2.10     
Glutamine synthetase root isozyme 1 (GS)   161G19T7   At1g66200   2.06     
Monosaccharide transporter STP3   AI997045   At5g61520   2.05     
Signal transduction (6)        
Calcium-dependent protein kinase   AI996555   At5g66210   2.96     
WD-40 repeat protein   AI993055   At5g14530   2.70   TTTCGCGG   −104   −  
Receptor-protein kinase-like protein   AI994727   At5g54380   2.59     
Putative phytochrome A   AI998146   At1g09570   2.45     
Putative leucine-rich receptor-like protein kinase   AI999651   At1g72180   2.13     
Putative receptor-like kinase   AI993298   At3g23750   2.06     
Others (13)        
Putative pollen allergen   AI996548   At3g45970   3.22     
Cold-regulated protein COR6,6   AW004198   At5g15970   3.03     
Phi-1-like protein   AI994601   At5g64260   2.60     
Lipid-transfer protein-like   AI998609   At5g01870   2.33     
DnaJ homologue   AI994551   At5g06910   2.32   ATTGGCGC   −103   +  
Blue copper-binding protein   AI996535   At5g20230   2.30     
Src-2-like protein   AI998679   Atlg09070   2.19     
Ring finger protein   AI999491   At3g61460   2.14     
Putative Tice22   AI993361   At3g23710   2.14     
Nodulin-like protein   AI996322   Atlg80530   2.07     
Putative resistance protein   AI997549   Atlg61100   2.06     
Seed imbitition protein-like   AI993446   At5g20250   2.05     
Putative disease resistance protein   AI998978   Atlg72900   2.04   TTTGGCGG   −175   +  
Unknown function (14)        
Putative protein   AI994686   At3g45730   5.14     
Putative protein   AI994734   At5g66580   3.18     
Unknown protein   AI999397   At2g38310   2.79   TTTGCCCC   −280   −  
Hypothetical protein   AI998042   Atlg57680   2.66     
Unknown protein   AI995465   At2g47440   2.50     
Unknown protein   AI994871   Atlg76970   2.34     
Hypothetical protein   AI998366   Atlg27500   2.21     
Putative protein   AI996967   At4g33050   2.20     
Putative protein   AI995917   At3g43690   2.18     
Unknown protein   AI993084   At2g25970   2.15     
Unknown protein   AI993077   Atlg68580   2.13     
Putative protein   AI993019   At5g14420   2.05   TTTCGCCG   −443   −  
Hypothetical protein   AI997428   Atlg57990   2.02     
Unknown protein   AI997827   At5g53740   2.01   TTTGGCGG   −66   +  
*

Relative position upstream from the translation initiation site.

Table 2.

Arabidopsis genes repressed twofold or more in E2Fa-DPa plants sorted according to functional category

Gene identification Accession number ORF name Fold repression E2F motif Position* Strand
Cell wall biogenesis (4)        
Similar to polygalacturonase-like protein   AI993509   At1g10640   3.62     
Putative xyloglucan endo-transglycosylase   AI997647   At2g36870   2.51     
Pectate lyase 1-like protein   AI994801   At1g67750   2.40     
Xyloglucan endo-transglycosylase   AI998832   At3g44990   2.35     
Metabolism and biogenesis (24)        
Fructose-bisphosphate aldolase-like protein   AI994456   At4g26530   5.99   ATTGGCCC   −426   −  
Sucrose-phosphate synthase-like protein   AI995432   At4g10120   4.64     
Putative branched-chain amino acid aminotransferase   AI997263   At3g19710   3.31     
Vitamin C-2   AI997404   At4g26850   3.04   TTTGCCGC   −222   +  
Nicotianamine synthase   AI993200   At5g04950   2.86     
β-fructosidase   AI994670   At1g62660   2.66   TTTCCCCC   −344   −  
Neoxanthin cleavage enzyme-like protein   AI997269   At4g19170   2.66     
Putative starch synthase   AI997174   At1g32900   2.63     
Cytochrome P450 monooxygenase (CYP83A1)   AI994017   At4g13770   2.57     
β-amylase-like protein   AI999322   At5g18670   2.53     
FRO1-like protein; NADPH oxidase-like   AI995987   At5g49740   2.46     
Putative hydrolase   AI997149   At3g48420   2.39     
Furamate hydratase   AI997067   At5g50950   2.31     
5′-adenylylsulfate reductase   AI992757   At1g62180   2.30     
5′-adenylylsulfate reductase   AI996614   At4g04610   2.30     
UDP rhamnose-anthocyanidin-3-glucoside rhamnosyltransferase-like protein   AI996803   At4g27560   2.24     
Cytochrome P450-like protein   AI993171   At5g48000   2.23     
Lactoylglutathione lyase-like protein   AI994552   At1g11840   2.20     
Putative β-glucosidase   AI995306   At4g27820   2.20   ATTGGCCC   −327   −  
Adenine phosphoribosyltransferase-like protein   AI994567   At4g22570   2.18     
Catalase   AI995830   At4g35090   2.17   ATTCCCCC   −168   +  
Putative glutathione peroxidase   AW004143   At2g25080   2.15     
Putative adenosine phosphosulfate kinase   AW004219   At2g14750   2.13     
Tyrosine transaminase-like protein   AI996914   At4g23600   2.13     
Transcription factors (5)        
Homeobox-leucine zipper protein ATHB-12   AI994027   At3g61890   4.20   ATTGGCCG   −113   −  
NAC domain protein NAC2   AI992865   At1g69490   3.68     
Myb-related transcription factor   AI995298   At1g71030   2.78     
Dof zinc finger protein   AI994875   At1g51700   2.30   TTTCCCGG   −18   +  
     TTTCCCCG   −13   −  
MYB-related transcription factor (CCA1)   AI992931   At2g46830   2.19     
Signal transduction (9)        
Serine/threonine protein kinase-like protein   AI995557   At5g10930   3.91     
Subtilisin proteinase-like   AI993428   At4g21650   3.19   TTTCGCGG   −85   +  
Putative oligopeptide transporter   AI996160   At4g10770   2.68     
Putative lectin   AI998542   At3g16400   2.52     
Ca2+-dependent membrane-binding protein annexin   AI998553   At1g35720   2.45     
Putative WD repeat protein   AI997238   At3g15880   2.38     
Putative lectin   AI999016   At3g16390   2.35     
Putative lectin   AI993358   At3g16530   2.31     
SNF1-related protein kinase (ATSRPK1)   AI993111   At3g23000   2.06     
Others (25)        
Putative protease inhibitor Dr4   AI995265   At1g73330   10.30     
Major latex protein homolog-like   AI998305   At2g01520   4.27     
Pollen allergen-like protein   AI993041   At1g24020   3.56   TTTGGCCG   −377   +  
Putative heat shock protein   AI997846   At1g06460   3.55     
Putative fibrillin   AI997199   At4g04020   3.55   TTTGGCCG   −435   −  
Major latex protein homolog-like   AI997255   At1g70890   3.50     
Putative nematode resistance protein   AI993740   At2g40000   2.95     
Putative auxin-regulated protein   AJ508998   At2g46690   2.86     
Putative myrosinase-binding protein   AI997583   At2g39310   2.61     
Ubiquitin-conjugating enzyme-like protein   AI997782   At5g56150   2.41     
Ubiquitin-conjugating enzyme E2-17 kDa 8   AI994771   At5g41700   2.40     
Vegetative storage protein Vsp2   AI999152   At5g24770   2.35     
Heat shock protein 70   AI994044   At3g12580   2.24     
Chloroplast outer envelope membrane protein   AI997015   At3g63160   2.20     
Translation initiation factor-like protein   AI992786   At5g54940   2.15     
Pseudogene   AI995323   At2g04110   2.07     
Vegetative storage protein Vsp 1   AI999546   At5g24780   2.06     
Dehydrin ERD10   AI997518   At1g20450   2.06     
MTN3-like protein   AI997159   At3g48740   2.05     
Putative chlorophyll a/b-binding protein   AI994859   At3g27690   2.05     
Photosystem I reaction center subunit psaN   AI997939   At5g64040   2.03     
Others - continued       
AR781 similar to yeast pheromone receptor   AI1998194   At2g26530   2.03     
Putative lipid transfer protein   AI997024   At2g15050   2.03     
Peroxidase ATP3a   AI998372   At5g64100   2.03   TTTGGCCG   −492   +  
Myosin heavy chain-like protein   AI999224   At3g16000   2.01     
Unknown function (35)        
Unknown protein   AI993767   At1g45200   3.91     
Putative protein   AI993468   At3g56290   3.38     
Hypothetical protein   AI996374   At1g61890   2.78     
Unknown protein   AI994573   At3g15950   2.71     
Putative protein   AI994726   At3g52360   2.65     
Hypothetical protein   AI997393   At4g02920   2.60   TTTGCCCC   −419   −  
Unknown protein   AJ508997   At5g43580   2.58     
Unknown protein   AI997866   At1g70760   2.52     
Unknown protein   AI997085   At5g43750   2.51     
Putative protein   AI995724   At5g50100   2.48     
Unknown protein   AI995337   At1g74880   2.42     
Unknown protein   AI998296   At3g19370   2.40     
Unknown protein   AI993346   At3g10420   2.40     
Putative protein   AI999485   At3g61080   2.38     
Unknown protein   AI996923   At1g67860   2.38     
Unknown protein   AI994841   At1g52870   2.35   ATTCCCCC   −74   +  
Unknown protein   AI999581   At1g64370   2.35     
Unknown protein   AI997584   At1g05870   2.25     
Putative protein   AI992938   At5g03540   2.21     
Hypothetical protein   AI997712   At2g15020   2.21     
Unknown protein   AI998338   At1g68440   2.20     
Unknown protein   AI996872   At2g21960   2.19     
Putative protein   AI996295   At4g27280   2.18     
Putative protein   AI995642   At3g48200   2.16     
Unknown protein   AI997470   At2g32870   2.14     
Hypothetical protein   AI998460   At1g69510   2.11   ATTCGCGG   −120   +  
     TTTGGCCC   −492   +  
Putative protein   AI993356   At5g22460   2.10     
Putative protein   AI995956   At5g52060   2.08     
Unknown protein   AI996100   At2g35830   2.06     
Hypothetical protein   AI996039   At3g27050   2.05   ATTGCCCC   −5   −  
Unknown protein   AI996020   At5g51720   2.04     
Putative protein   AW004101   At4g39730   2.03     
Hypothetical protein   AI998372   At2g01260   2.03     
Unknown protein   AI999573   At3g61060   2.00     
Unknown protein   AI998562   At2g35760   2.00     
No hit (2)        
No hit on genome   AI995690    2.54     
No hit on genome   AI999010    2.23     
Gene identification Accession number ORF name Fold repression E2F motif Position* Strand
Cell wall biogenesis (4)        
Similar to polygalacturonase-like protein   AI993509   At1g10640   3.62     
Putative xyloglucan endo-transglycosylase   AI997647   At2g36870   2.51     
Pectate lyase 1-like protein   AI994801   At1g67750   2.40     
Xyloglucan endo-transglycosylase   AI998832   At3g44990   2.35     
Metabolism and biogenesis (24)        
Fructose-bisphosphate aldolase-like protein   AI994456   At4g26530   5.99   ATTGGCCC   −426   −  
Sucrose-phosphate synthase-like protein   AI995432   At4g10120   4.64     
Putative branched-chain amino acid aminotransferase   AI997263   At3g19710   3.31     
Vitamin C-2   AI997404   At4g26850   3.04   TTTGCCGC   −222   +  
Nicotianamine synthase   AI993200   At5g04950   2.86     
β-fructosidase   AI994670   At1g62660   2.66   TTTCCCCC   −344   −  
Neoxanthin cleavage enzyme-like protein   AI997269   At4g19170   2.66     
Putative starch synthase   AI997174   At1g32900   2.63     
Cytochrome P450 monooxygenase (CYP83A1)   AI994017   At4g13770   2.57     
β-amylase-like protein   AI999322   At5g18670   2.53     
FRO1-like protein; NADPH oxidase-like   AI995987   At5g49740   2.46     
Putative hydrolase   AI997149   At3g48420   2.39     
Furamate hydratase   AI997067   At5g50950   2.31     
5′-adenylylsulfate reductase   AI992757   At1g62180   2.30     
5′-adenylylsulfate reductase   AI996614   At4g04610   2.30     
UDP rhamnose-anthocyanidin-3-glucoside rhamnosyltransferase-like protein   AI996803   At4g27560   2.24     
Cytochrome P450-like protein   AI993171   At5g48000   2.23     
Lactoylglutathione lyase-like protein   AI994552   At1g11840   2.20     
Putative β-glucosidase   AI995306   At4g27820   2.20   ATTGGCCC   −327   −  
Adenine phosphoribosyltransferase-like protein   AI994567   At4g22570   2.18     
Catalase   AI995830   At4g35090   2.17   ATTCCCCC   −168   +  
Putative glutathione peroxidase   AW004143   At2g25080   2.15     
Putative adenosine phosphosulfate kinase   AW004219   At2g14750   2.13     
Tyrosine transaminase-like protein   AI996914   At4g23600   2.13     
Transcription factors (5)        
Homeobox-leucine zipper protein ATHB-12   AI994027   At3g61890   4.20   ATTGGCCG   −113   −  
NAC domain protein NAC2   AI992865   At1g69490   3.68     
Myb-related transcription factor   AI995298   At1g71030   2.78     
Dof zinc finger protein   AI994875   At1g51700   2.30   TTTCCCGG   −18   +  
     TTTCCCCG   −13   −  
MYB-related transcription factor (CCA1)   AI992931   At2g46830   2.19     
Signal transduction (9)        
Serine/threonine protein kinase-like protein   AI995557   At5g10930   3.91     
Subtilisin proteinase-like   AI993428   At4g21650   3.19   TTTCGCGG   −85   +  
Putative oligopeptide transporter   AI996160   At4g10770   2.68     
Putative lectin   AI998542   At3g16400   2.52     
Ca2+-dependent membrane-binding protein annexin   AI998553   At1g35720   2.45     
Putative WD repeat protein   AI997238   At3g15880   2.38     
Putative lectin   AI999016   At3g16390   2.35     
Putative lectin   AI993358   At3g16530   2.31     
SNF1-related protein kinase (ATSRPK1)   AI993111   At3g23000   2.06     
Others (25)        
Putative protease inhibitor Dr4   AI995265   At1g73330   10.30     
Major latex protein homolog-like   AI998305   At2g01520   4.27     
Pollen allergen-like protein   AI993041   At1g24020   3.56   TTTGGCCG   −377   +  
Putative heat shock protein   AI997846   At1g06460   3.55     
Putative fibrillin   AI997199   At4g04020   3.55   TTTGGCCG   −435   −  
Major latex protein homolog-like   AI997255   At1g70890   3.50     
Putative nematode resistance protein   AI993740   At2g40000   2.95     
Putative auxin-regulated protein   AJ508998   At2g46690   2.86     
Putative myrosinase-binding protein   AI997583   At2g39310   2.61     
Ubiquitin-conjugating enzyme-like protein   AI997782   At5g56150   2.41     
Ubiquitin-conjugating enzyme E2-17 kDa 8   AI994771   At5g41700   2.40     
Vegetative storage protein Vsp2   AI999152   At5g24770   2.35     
Heat shock protein 70   AI994044   At3g12580   2.24     
Chloroplast outer envelope membrane protein   AI997015   At3g63160   2.20     
Translation initiation factor-like protein   AI992786   At5g54940   2.15     
Pseudogene   AI995323   At2g04110   2.07     
Vegetative storage protein Vsp 1   AI999546   At5g24780   2.06     
Dehydrin ERD10   AI997518   At1g20450   2.06     
MTN3-like protein   AI997159   At3g48740   2.05     
Putative chlorophyll a/b-binding protein   AI994859   At3g27690   2.05     
Photosystem I reaction center subunit psaN   AI997939   At5g64040   2.03     
Others - continued       
AR781 similar to yeast pheromone receptor   AI1998194   At2g26530   2.03     
Putative lipid transfer protein   AI997024   At2g15050   2.03     
Peroxidase ATP3a   AI998372   At5g64100   2.03   TTTGGCCG   −492   +  
Myosin heavy chain-like protein   AI999224   At3g16000   2.01     
Unknown function (35)        
Unknown protein   AI993767   At1g45200   3.91     
Putative protein   AI993468   At3g56290   3.38     
Hypothetical protein   AI996374   At1g61890   2.78     
Unknown protein   AI994573   At3g15950   2.71     
Putative protein   AI994726   At3g52360   2.65     
Hypothetical protein   AI997393   At4g02920   2.60   TTTGCCCC   −419   −  
Unknown protein   AJ508997   At5g43580   2.58     
Unknown protein   AI997866   At1g70760   2.52     
Unknown protein   AI997085   At5g43750   2.51     
Putative protein   AI995724   At5g50100   2.48     
Unknown protein   AI995337   At1g74880   2.42     
Unknown protein   AI998296   At3g19370   2.40     
Unknown protein   AI993346   At3g10420   2.40     
Putative protein   AI999485   At3g61080   2.38     
Unknown protein   AI996923   At1g67860   2.38     
Unknown protein   AI994841   At1g52870   2.35   ATTCCCCC   −74   +  
Unknown protein   AI999581   At1g64370   2.35     
Unknown protein   AI997584   At1g05870   2.25     
Putative protein   AI992938   At5g03540   2.21     
Hypothetical protein   AI997712   At2g15020   2.21     
Unknown protein   AI998338   At1g68440   2.20     
Unknown protein   AI996872   At2g21960   2.19     
Putative protein   AI996295   At4g27280   2.18     
Putative protein   AI995642   At3g48200   2.16     
Unknown protein   AI997470   At2g32870   2.14     
Hypothetical protein   AI998460   At1g69510   2.11   ATTCGCGG   −120   +  
     TTTGGCCC   −492   +  
Putative protein   AI993356   At5g22460   2.10     
Putative protein   AI995956   At5g52060   2.08     
Unknown protein   AI996100   At2g35830   2.06     
Hypothetical protein   AI996039   At3g27050   2.05   ATTGCCCC   −5   −  
Unknown protein   AI996020   At5g51720   2.04     
Putative protein   AW004101   At4g39730   2.03     
Hypothetical protein   AI998372   At2g01260   2.03     
Unknown protein   AI999573   At3g61060   2.00     
Unknown protein   AI998562   At2g35760   2.00     
No hit (2)        
No hit on genome   AI995690    2.54     
No hit on genome   AI999010    2.23     
*

Relative position upstream from the translation initiation site.

In addition to the overexpressed E2Fa gene (90-fold more abundant in transgenic than in control plants), only one cell cycle gene (CDKB1;1) has a twofold or more change in expression level upon E2Fa-DPa overexpression. CDKB1;1 had already been predicted to be a candidate E2F-DP target by the presence of a consensus E2F-DP-binding site in its promoter (de Jager et al., 2001). Whereas CDKB1;1 activity is highest at the G2 to M transition, its transcript levels start to increase during S phase (Porceddu et al., 1999; Menges and Murray, 2002). Therefore, up-regulation of CDKB1;1 might be a mechanism linking DNA replication with the following mitosis. That other cell cycle genes modulated in the E2Fa-DPa plants are not detected can be explained by the lack of many important E2F-DP target genes on the microarray and the putative difficulty in identifying changes in expression levels of lowly expressed genes in microarray hybridizations.

Cell wall biogenesis genes

Four members of the xyloglucan endotransglucosylase (XET) gene family are found to be twofold or more up-regulated in the E2Fa-DPa plants, one of them identical to the previously described Meri-5 gene (Medford et al., 1991). XETs are enzymes that modify cell wall components and are presumed to play a role in altering size, shape and physical properties of plant cells. Reversal breakage of the xyloglucan tethers by XETs has been proposed as a mechanism for allowing cell wall loosening in turgor-driven cell expansion (Campbell and Braam, 1999). However, there are several reasons for believing that E2Fa-DPa-induced XETs are not required for cell expansion. First, cells divide more frequently in the E2Fa-DPa plants, but the overall cell size is smaller in transgenic than in control plants; so, no overall increase in expansion rates is needed. Second, no induction is seen of genes with a known role in cell expansion, such as expansins. Therefore, the hydrolytic activity of the XETs might rather be required to incorporate the newly synthesized cell walls formed during cytokinesis into the existing cell wall structure. Alternatively, because XET activity has been shown to be involved in the postgerminative mobilization of xyloglucan storage reserves in Nasturtium cotyledons (Farkas et al., 1992; Fanutti et al., 1993), induction of XETs in E2Fa-DPa plants might be related to polysaccharide breakdown to serve the metabolic and energy needs that are required to synthesize new nucleotides (see below).

Interestingly, two XETs can be identified in the set of twofold-or-more down-regulated genes. These XETs are more related to each other than to the induced XET genes. This differential response of XETs toward the E2Fa-DPa-induced phenotypes suggests that plant XETs can be classified into at least two different functional classes.

Genes involved in metabolism and biogenesis

A relatively large number of genes involved in metabolism and biogenesis were found in both the up-regulated and down-regulated gene groups. Most remarkable is the induction of genes involved in nitrogen assimilation, such as nitrate reductase (NIA2), glutamine synthetase (GS), and glutamate synthase (GOGAT) (Fig. 3). Although not present on the microarray, the nitrite reductase (NiR) gene was found to be induced as well in the transgenic lines, as demonstrated by RT-mediated PCR analysis (Fig. 2). Nitrogen and nitrite reductase catalyze the first two steps in the nitrogen assimilation pathway, whereas GS and GOGAT are involved both in the primary assimilation of nitrogen and the reassimilation of free ammonium. This mechanism supplies the plant with all nitrogen needed for the biosynthesis of amino acids and other nitrogen-containing compounds.

Fig. 3.

Sources of α-ketoglutarate in the E2Fa-DPa-overproducing cells. Genes encoding enzymes shown in red and green are up-regulated or down-regulated in the E2Fa-DPa versus wild-type plants, respectively. Products indicated in blue act as precursors for nucleotide biosynthesis. α-KG, α-ketoglutarate; GOGAT, glutamate synthase; GS, glutamine synthetase; NIA2, nitrate reductase; NiR, nitrite reductase.

Fig. 3.

Sources of α-ketoglutarate in the E2Fa-DPa-overproducing cells. Genes encoding enzymes shown in red and green are up-regulated or down-regulated in the E2Fa-DPa versus wild-type plants, respectively. Products indicated in blue act as precursors for nucleotide biosynthesis. α-KG, α-ketoglutarate; GOGAT, glutamate synthase; GS, glutamine synthetase; NIA2, nitrate reductase; NiR, nitrite reductase.

There are other indications that the nitrogen metabolism is altered in the E2Fa-DPa plants; these include the modification of genes homologous to genes expressed during the formation of nitrogen-fixing nodules in Medicago sativa (MTN3 and a nodulin-like gene), and the down-regulation of genes involved in sulfur assimilation (two different genes encoding adenylylsulfate reductase [APR] and a putative adenine phosphosulfate kinase). Genes involved in sulfur assimilation have been shown before to be transcriptionally down-regulated during nitrogen deficiency (Koprikova et al., 2000).

The altered expression of genes involved in nitrogen assimilation and metabolism in the E2Fa-DPa transgenic plants might reflect the need for nitrogen for the nucleotide biosynthesis, because purine and pyrimidine bases are rich in nitrogen. If nitrogen assimilation were indeed stimulated by E2Fa-DPa overexpression, two requirement should be fulfilled. Firstly, there should be enough α-ketoglutarate to act as an acceptor molecule for ammonium (Lancien et al., 2000) and secondly, because assimilation of nitrogen is energy consuming, the rate of reductant production should be higher in the E2Fa-DPa transgenic than in the wild-type plants.

Our microarray data suggest that in the accumulation of α-ketoglutarate in E2Fa-DPa-overexpressing plants is stimulated in different ways. First, α-ketoglutarate production is improved by increased photosynthetic activity, as indicated by the 4.7-fold up-regulation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Fig. 3), with accumulation of glyceraldehyde-3-phosphate as a result. Glyceraldehyde-3-phosphate can be converted into fructose-1,6-bisphosphate by fructose bisphosphate aldolase. However, a sixfold down-regulation of the fructose bisphosphate aldolase gene rather suggests the conversion of glyceraldehyde-3-phosphate into pyruvate, which can be converted into α-ketoglutarate in the citrate cycle. The preferential conversion of glyceraldehyde-3-phosphate to pyruvate fits the increased need for amino acids rather than for sugars to drive nucleotide biosynthesis (Fig. 3).

A second source of α-ketoglutarate can be provided by the glyoxylate cycle. In E2Fa-DPa-overproducing plants we observed a 3.1-fold increase in expression of isocitrate lyase, suggesting an increased lipid turnover. Isocitrate lyase activity cleaves isocitrate into glyoxylate and succinate (Fig. 3). Whereas the produced glyoxylate can be converted into glycine, which is also required for de novo nucleotide biosynthesis, succinate can be converted into α-ketoglutarate in the citrate cycle. A 2.3-fold decrease in the expression of the fumarase gene presumably stimulates the subsequent conversion of α-ketoglutarate to glutamate by triggering an accumulation of succinate and fumarate, which are also side products formed during de novo nucleotide biosynthesis (Fig. 3). Reductant in plants mainly originates from photosynthetic electron transport in leaves. Corresponding with the increased need for reductant, several components of the chloroplast electron transport chain and associated ATP-synthesizing apparatus, such as cytochrome B6, a photosystem II subunit, and the ATPase ϵ subunit, are up-regulated in the E2Fa-DPa transgenic plants. Increased expression of the protochlorophyllide reductase precursor even indicates an increase in chlorophyll biosynthesis.

E2Fa-DPa plants may suffer from nitrogen starvation that has an impact on amino acid biosynthesis. Three different amino acid aminotransferases are down-regulated in the E2Fa-DPa plants. Shortage of nitrogen-rich amino acids is also evident from the reduced expression of genes encoding vegetative storage proteins (VSP1 and VSP2) and ERD10, a protein with a compositional bias toward glutamate (Kiyosue et al., 1994). Additional evidence for amino acid shortage comes from the down-regulation of a myrosinase-binding protein and the cytochrome P450 monooxygenase CYP83A1. Both proteins are involved in the biosynthesis of glucosinolates, nitrogen- and sulfur-containing products derived from amino acids (Wittstock and Halkier, 2002).

Promoter analysis of E2Fa-DPa-regulated genes

The DNA-binding domains of the E2F and DP proteins are highly conserved between plants and mammals and, correspondingly, plant E2F-DP proteins have been shown by the technique of electrophoresis mobility shift assay to bind to the same canonical DNA-binding site as their mammalian counterparts (Albani et al., 2000; Ramirez-Parra and Gutierrez, 2000; de Jager et al., 2001). Furthermore, these E2F-binding sites regulate the expression of several plant genes involved in DNA synthesis (Kosugi and Ohashi, 2002a; Chabouté et al., 2000; Castellano et al., 2001; Egelkrout et al., 2001; Stevens et al., 2002).

To distinguish between the putatively direct target genes of E2Fa-DPa and the secondarily induced genes, the first 500 bp upstream of the ATG start codon of the genes with 2-fold or higher change in expression were scanned for the presence of an E2F-like-binding site matching the (A/T)TT(G/C)(G/C)C(G/C)(G/C) sequence, which corresponds to all the different E2F-DP-binding motifs that have been described in plants. Of all the different permutations only the TTTCCCGC and TTTGGCGG elements were enriched significantly (P<0.01) in the set of E2Fa-DPaupregulated genes, suggesting these are the preferred binding site of the E2Fa-DPa complex (Table 3). Moreover, six out of eight target genes containing one or more of these elements belong to the group of genes involved in DNA replication and modification. The observation that not all genes that enclase this DNA sequence in their promoter are induced upon E2Fa-DPa overexpression suggests that the presence of the TTTCCCGC or TTTGGCGG motif is not the only element to make a gene responsive toward E2Fa-DPa, and that E2Fa-DPa may cooperate with other factors to activate transcription. Alternatively, the promoters of non-responsive genes might be shielded with other transcription factor complexes. A putative candidate is the E2Fc protein which, in analogy with the mammalian E2F6 protein, lacks a strong transactivation domain (del Pozo et al., 2002). Alternative candidates are the recently discovered DEL proteins, proven to bind as monomers to the canonical E2F-binding site (Kosugi and Ohashi, 2002b; Mariconti et al., 2002). Because of a lack of transcriptional activation domain, the DEL proteins are postulated to act as repressors of E2F-DP-regulated genes by competing for the same binding site.

Table 3.

Number of E2F elements in the different data sets

E2F motif All genes (4390)* Upregulated genes (84) Downregulated genes (104)
TTTCCCCC   49   2   1  
TTTCCCCG   31   1   1  
TTTCCCGC   46   5   0  
TTTCCCGG   61   1   1  
TTTCGCCC   16   0   0  
TTTCGCCG   76   2   0  
TTTCGCGC   19   2   0  
TTTCGCGG   30   2   1  
TTTGCCCC   35   1   1  
TTTGCCCG   13   0   0  
TTTGCCGC   24   0   1  
TTTGCCGG   34   0   0  
TTTGGCCC   54   0   1  
TTTGGCCG   38   0   3  
TTTGGCGC   18   1   0  
TTTGGCGG   47   4   0  
ATTCCCCC   14   0   2  
ATTCCCCG   21   0   0  
ATTCCCGC   11   1   0  
ATTCCCGG   23   0   0  
ATTCGCCC   10   0   0  
ATTCGCCG   42   0   0  
ATTCGCGC   13   1   0  
ATTCGCGG   9   0   1  
ATTGCCCC   14   0   1  
ATTGCCCG   6   0   0  
ATTGCCGC   15   0   0  
ATTGCCGG   0   0   0  
ATTGGCCC   42   0   2  
ATTGGCCG   13   0   1  
ATTGGCGC   12   2   0  
ATTGGCGG   28   0   0  
Total   864   25   17  
E2F motif All genes (4390)* Upregulated genes (84) Downregulated genes (104)
TTTCCCCC   49   2   1  
TTTCCCCG   31   1   1  
TTTCCCGC   46   5   0  
TTTCCCGG   61   1   1  
TTTCGCCC   16   0   0  
TTTCGCCG   76   2   0  
TTTCGCGC   19   2   0  
TTTCGCGG   30   2   1  
TTTGCCCC   35   1   1  
TTTGCCCG   13   0   0  
TTTGCCGC   24   0   1  
TTTGCCGG   34   0   0  
TTTGGCCC   54   0   1  
TTTGGCCG   38   0   3  
TTTGGCGC   18   1   0  
TTTGGCGG   47   4   0  
ATTCCCCC   14   0   2  
ATTCCCCG   21   0   0  
ATTCCCGC   11   1   0  
ATTCCCGG   23   0   0  
ATTCGCCC   10   0   0  
ATTCGCCG   42   0   0  
ATTCGCGC   13   1   0  
ATTCGCGG   9   0   1  
ATTGCCCC   14   0   1  
ATTGCCCG   6   0   0  
ATTGCCGC   15   0   0  
ATTGCCGG   0   0   0  
ATTGGCCC   42   0   2  
ATTGGCCG   13   0   1  
ATTGGCGC   12   2   0  
ATTGGCGG   28   0   0  
Total   864   25   17  
*

Promoters of mitochondrial and chloroplastic genes were omitted from this analysis

It is not excluded that genes without an E2F-like-binding site are not directly activated by E2Fa-DPa. Chromatin immunoprecipitation experiments have shown that mammalian E2F factors can bind to promoters without a clear E2F recognition motif (Kiyosue et al., 1994), suggesting that E2FDP might recognize non-canonical binding sites, or might be recruited by promoters through the association of other factors. In this respect, the Chlorella vulgaris nitrate reductase gene, of which the Arabidopsis homologue was shown here to be induced by E2F-DPa, binds an E2F-DP complex, although a clear consensus binding site is lacking (Cannons and Shiflett, 2001).

E2Fs can activate as well as repress promoter activity (Trimarchi and Lees, 2002). In the PCNA, MCM3 and RNR2 promoters, E2F sequences have been identified that act as a negative regulatory element during development (Chabouté et al., 2000; Egelkrout et al., 2001; Stevens et al., 2002). In the set of down-regulated genes, no particular enrichment of a specific E2F sequence could be seen (Table 3). Therefore, the data suggest that the E2Fa-DPa complex works as a transcriptional activator and that other E2F-DP complexes are involved in E2F-mediated transcriptional repression.

Conclusions

Microarray analysis of E2Fa-DPa-overexpressing lines identified a cross-talking genetic network between DNA replication, nitrogen assimilation and photosynthesis. The putatively direct E2Fa-DPa target genes as identified by the presence of an E2F-DP-binding site, belong to the group of genes involved in DNA synthesis, whereas the secondarily induced genes are mainly linked to nitrogen assimilation. In a recently published microarray experiment in which the periodic expression of genes during the cell cycle was monitored, genes with a role in nitrogen assimilation (aspartate aminotransferase and a nitrate transporter) were found to be specifically expressed during the S phase (Menges et al., 2002). Because purine and pyrimidine bases are nitrogen rich, we postulate that induction of nitrogen assimilation genes during DNA synthesis in wild-type and E2Fa-DPa transgenic plants is required to supply enough nitrogen for nucleotide biosynthesis. However, in the EFa-DPa transgenic plants, increased nitrogen assimilation most probably does not meet all the nucleotide biosynthesis needs, as seen by the expression modulation of many genes involved in nitrogen and carbohydrate metabolism. The drain of nitrogen from essential biosynthetic pathways to the nucleotide biosynthesis pathway is expected to affect other aspects of plant metabolism, as can be seen from the reduced expression of vegetative storage protein genes and genes involved in amino acid biosynthesis. This altered metabolism might, at least in part, contribute to the growth arrest observed in E2Fa-DPa transgenic plants.

The exact regulatory pathways and factors controlling the nitrogen assimilation pathway in plants are still unknown. In addition to the genes involved in DNA replication and metabolism, our data contain a relatively large number of genes with unspecified function (Tables 1 and 2). For instance, a GATA zinc-finger-encoded gene with a still unknown function is found between the up-regulated regulatory genes. This gene might encode the ortholog of the Neurospora crassa nit-2 protein that has been shown to positively regulate expression of the nitrate reductase gene (Fu and Marzluf, 1990). Other regulatory genes modified in the E2Fa-DPa plants encode protein kinases and several putative receptor kinases. These genes might include some novel key regulatory components in the process of nitrogen assimilation or regulation of efficient nitrogen usage. It will be of great interest to analyze their role in nitrogen assimilation, metabolism and plant growth.

Acknowledgements

The authors thank the members of the cell cycle group for fruitful discussions and useful suggestions and Martine De Cock for help in preparing the manuscript. This work was supported by grants from the Interuniversity Poles of Attraction Programme (Belgian State, Prime Minister's Office - Federal Office for Scientific, Technical and Cultural Affairs; P5/13), the European Union (ECCO QLG2-CT1999-00454), CropDesign NV (0235), and the Fund for Scientific Research (Flanders) (G.0025.02). K.V. and K.F. are indebted to the Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen for predoctoral fellowships. L.D.V. is a postdoctoral fellow of the Fund for Scientific Research (Flanders).

References

Albani, D., Mariconti, L., Ricagno, S., Pitto, L., Moroni, C., Helin, K. and Cella, R. (
2000
). DcE2F, a functional plant E2F-like transcriptional activator from Daucus carota.
J. Biol. Chem.
275
,
19258
-19267.
Campbell, P. and Braam, J. (
1999
). Xyloglucan endotransglycosylases: diversity of genes, enzymes and potential wall-modifying functions.
Trends Plant Sci.
4
,
361
-366.
Cannons, A. C. and Shiflett, S. D. (
2001
). Transcriptional regulation of the nitrate reductase gene in Chlorella vulgaris: identification of regulatory elements controlling expression.
Curr. Genet.
40
,
128
-135.
Castellano, M. M., del Pozo, J. C., Ramirez-Parra, E., Brown, S. and Gutierrez, C. (
2001
). Expression and stability of Arabidopsis CDC6 are associated with endoreplication.
Plant Cell
13
,
2671
-2686.
Chabouté, M.-E., Clément, B., Sekine, M., Philipps, G. and Chaubet-Gigot, N. (
2000
). Cell cycle regulation of the tobacco ribonucleotide reductase small subunit gene is mediated by E2F-like elements.
Plant Cell
12
,
1987
-1999.
de Jager, S. M., Menges, M., Bauer, U.-M. and Murray, J. A. H. (
2001
). Arabidopsis E2F1 binds a sequence present in the promoter of S-phase-regulated gene AtCDC6 and is a member of a multigene family with differential activities.
Plant Mol. Biol.
47
,
555
-568.
De Veylder, L., Beeckman, T., Beemster, G. T. S., de Almeida Engler, J., Ormenese, S., Maes, S., Naudts, M., Van Der Schueren, E., Jacqmard, A., Engler, G. and Inzé, D. (
2002
). Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa/DPa transcription factor.
EMBO J.
21
,
1360
-1368.
del Pozo, J. C., Boniotti, M. B. and Gutierrez, C. (
2002
). Arabidopsis E2Fc functions in cell division and is degraded by the ubiquitin-SCFAtSKP2 pathway in response to light.
Plant Cell
14
,
3057
-3071.
Egelkrout, E. M., Robertson, D. and Hanley-Bowdoin, L. (
2001
). Proliferating cell nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves.
Plant Cell
13
,
1437
-1452.
Fanutti, C., Gidley, M. J. and Reid, J. S. G. (
1993
). Action of a pure xyloglucan endo-transglycosylase (formerly called xyloglucan-specific endo-(1→4)-β-d-glucanase) from the cotyledons of germinated nasturtium seeds.
Plant J.
3
,
691
-700.
Farkas, V., Sulova, Z., Stratilova, E., Hanna, R. and Maclachlan, G. (
1992
). Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides.
Arch. Biochem. Biophys.
298
,
365
-370.
Fu, Y.-H. and Marzluf, G. A. (
1990
). nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein.
Proc. Natl. Acad. Sci. USA
87
,
5331
-5335.
Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M. and Nevins, J. R. (
2001
). Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis.
Mol. Cell. Biol.
21
,
4684
-4699.
Kel, A. E., Kel-Margoulis, O. V., Farnham, P. J., Bartley, S. M., Wingender, E. and Zhang, M. Q. (
2001
). Computer-assisted identification of cell cycle-related genes: new targets for E2F transcription factors.
J. Mol. Biol.
309
,
99
-120.
Kiyosue, T., Yamaguchi-Shinozaki, K. and Shinozaki, K. (
1994
). Cloning of cDNAs for genes that are early-responsive to dehydration stress (ERDs) in Arabidopsis thaliana L.: identification of three ERDs as HSP cognate genes.
Plant Mol. Biol.
25
,
791
-798.
Koprivova, A., Suter, M., Op den Camp, R., Brunold, C. and Kopriva, S. (
2000
). Regulation of sulfate assimilation by nitrogen in Arabidopsis.
Plant Physiol.
122
,
737
-746.
Kosugi, S. and Ohashi, Y. (
2002a
). E2F sites that can interact with E2F proteins cloned from rice are required for meristematic tissue-specific expression of rice and tobacco proliferating cell nuclear antigen promoters.
Plant J.
29
,
45
-59.
Kosugi, S. and Ohashi, Y. (
2002b
). E2Ls, E2F-like repressors of Arabidopsis that bind to E2F sites in a monomeric form.
J. Biol. Chem.
277
,
16553
-16558.
Lancien, M., Gadal, P. and Hodges, M. (
2000
). Enzyme redundancy and the importance of 2-oxoglutarate in higher plant ammonium assimilation.
Plant Physiol.
123
,
817
-824.
Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rouzé, P. and Rombauts, S. (
2002
). PlantCare: a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences.
Nucleic Acids Res.
30
,
325
-327.
Lusser, A., Eberharter, A., Loidl, A., Goralik-Schramel, M., Horngacher, M., Haas, H. and Loidl, P. (
1999
). Analysis of the histone acetyltransferase B complex of maize embryos.
Nucleic Acids Res.
27
,
4427
-4435.
Magyar, Z., Atanassova, A., De Veylder, L., Rombauts, S. and Inzé, D. (
2000
). Characterization of two distinct DP-related genes from Arabidopsis thaliana.
FEBS Lett.
486
,
79
-87.
Mariconti, L., Pellegrini, B., Cantoni, R., Stevens, R., Bergounioux, C., Cella, R. and Albani, D. (
2002
). The E2F family of transcription factors from Arabidopsis thaliana. Novel and conserved components of the retinoblastoma/E2F pathway in plants.
J. Biol. Chem.
277
,
9911
-9919.
Medford, J. I., Elmer, J. S. and Klee, H. J. (
1991
). Molecular cloning and characterization of genes expressed in shoot apical meristems.
Plant Cell
3
,
359
-370.
Menges, M. and Murray, J. A. H. (
2002
). Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity.
Plant J.
20
,
203
-212.
Menges, M., Hennig, L., Gruissem, W. and Murray, J. A. H. (
2002
). Cell cycle-regulated gene expression in Arabidopsis.
J. Biol. Chem.
277
,
41987
-42002.
Müller, H., Bracken, A. P., Vernell, R., Moroni, M. C., Christians, F., Grassilli, E., Prosperini, E., Vigo, E., Oliner, J. D. and Helin, K. (
2001
). E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis.
Genes Dev.
15
,
267
-285.
Porceddu, A., De Veylder, L., Hayles, J., Van Montagu, M., Inzé, D. and Mironov, V. (
1999
). Mutational analysis of two Arabidopsis thaliana cyclin-dependent kinases in fission yeast.
FEBS Lett.
446
,
182
-188.
Puskás, L. G., Zvara, A., Hackler, L. J. and Van Hummelen, P. (
2002
). RNA amplification results in reproducible microarray data with slight ratio bias.
Biotechniques
32
,
1330
-1341.
Ramirez-Parra, E. and Gutierrez, C. (
2000
). Characterization of wheat DP, a heterodimerization partner of the plant E2F transcription factor which stimulates E2F-DNA binding.
FEBS Lett.
486
,
73
-78.
Ramirez-Parra, E., Fründt, C. and Gutierrez, C. (
2003
). A genome-wide identification of E2F-regulated genes in Arabidopsis.
Plant J.
33
,
801
-811.
Ramírez-Parra, E., Xie, Q., Boniotti, M. B. and Gutierrez, C. (
1999
). The cloning of plant E2F, a retinoblastoma-binding protein, reveals unique and conserved features with animal G1/S regulators.
Nucleic Acids Res.
27
,
3527
-3533.
Ren, B., Cam, H., Takahashi, Y., Volkert, T., Terragni, J., Young, R. A. and Dynlacht, B. D. (
2002
). E2F integrates cell cycle progression with DNA repair, replication, and G2/M checkpoints.
Genes Dev.
16
,
245
-256.
Sekine, M., Ito, M., Uemukai, K., Maeda, Y., Nakagami, H. and Shinmyo, A. (
1999
). Isolation and characterization of the E2F-like gene in plants.
FEBS Lett.
460
,
117
-122.
Stevens, R., Mariconti, L., Rossignol, P., Perennes, C., Cella, R. and Bergounioux, C. (
2002
). Two E2F sites in the Arabidopsis MCM3 promoter have different roles in cell cycle activation and meristematic expression.
J. Biol. Chem.
277
,
32978
-32984.
Trimarchi, J. M. and Lees, J. A. (
2002
). Sibling rivalry in the E2F family.
Nature Rev. Mol. Cell. Biol.
3
,
11
-20.
Valvekens, D., Van Montagu, M. and Van Lijsebettens, M. (
1988
). Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection.
Proc. Natl. Acad. Sci. USA
85
,
5536
-5540.
Vandepoele, K., Raes, J., De Veylder, L., Rouzé, P., Rombauts, S. and Inzé, D. (
2002
). Genome-wide analysis of core cell cycle genes in Arabidopsis.
Plant Cell
14
,
903
-916.
Weinmann, A., Bartley, S. M., Zhang, T., Zhang, M. Q. and Farnham, P. J. (
2001
). Use of chromatin immunoprecipitation to clone novel E2F target promoters.
Mol. Cell. Biol.
21
,
6820
-6832.
Wittstock, U. and Halkier, B. A. (
2002
). Glucosinolate research in the Arabidopsis era.
Trends Plant Sci.
7
,
263
-270.
Wolfinger, R. D., Gibson, G., Wolfinger, E. D., Bennett, L., Hamadeh, H., Bushel, P., Afshari, C. and Paules, R. S. (
2001
). Assessing gene significance from cDNA microarray expression data via mixed models.
J. Comput. Biol.
8
,
625
-637.
Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J. and Speed, T. P. (
2002
). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.
Nucleic Acids Res.
30
,
e15
.