The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants

Abstract

Background: The maize INDETERMINATE1 gene, ID1, is a key regulator of the transition to flowering and the founding member of a transcription factor gene family that encodes a protein with a distinct arrangement of zinc finger motifs. The zinc fingers and surrounding sequence make up the signature ID domain (IDD), which appears to be found in all higher plant genomes. The presence of zinc finger domains and previous biochemical studies showing that ID1 binds to DNA suggests that members of this gene family are involved in transcriptional regulation.

Results: Comparison of IDD genes identified in Arabidopsis and rice genomes, and all IDD genes discovered in maize EST and genomic databases, suggest that ID1 is a unique member of this gene family. High levels of sequence similarity amongst all IDD genes from maize, rice and Arabidopsis suggest that they are derived from a common ancestor. Several unique features of ID1 suggest that it is a divergent member of the maize IDD family. Although no clear ID1 ortholog was identified in the Arabidopsis genome, highly similar genes that encode proteins with identity extending beyond the ID domain were isolated from rice and sorghum. Phylogenetic comparisons show that these putative orthologs, along with maize ID1, form a group separate from other IDD genes. In contrast to ID1 mRNA, which is detected exclusively in immature leaves, several maize IDD genes showed a broad range of expression in various tissues. Further, Western analysis with an antibody that cross-reacts with ID1 protein and potential orthologs from rice and sorghum shows that all three proteins are detected in immature leaves only.

Conclusion: Comparative genomic analysis shows that the IDD zinc finger family is highly conserved among both monocots and dicots. The leaf-specific ID1 expression pattern distinguishes it from other maize IDD genes examined. A similar leaf-specific localization pattern was observed for the putative ID1 protein orthologs from rice and sorghum. These similarities between ID1 and closely related genes in other grasses point to possible similarities in function.

Figure 1

Gene structure of IDD genes from maize, rice and Arabidopsis and SbID from sorghum. Exons are represented by the open boxes and introns by black lines between boxes. The putative NLS is shown by a dark bar in the first exon; each zinc finger is shown by facing colored triangles (ZF1, orange; ZF2, blue; ZF3, green and ZF4, red). The amino acid spacer between ZF1 and ZF2 is represented by a dotted line. Numbers represent intron size; numbers between forward slashes signify exon size. Introns sizes preceded by '>' indicate that the full sequence is not known but that it is at least as long as indicated. The C-terminal conserved motifs, MSATALLQKAA (light blue box) and TR/LDFLG (purple box) are in the last exon. The asterisk (*) indicates that a stop codon could not be identified. Z = ZmIDD, O = OsIDD, A = AtIDD.

Figure 2

Alignment of deduced ID-domain amino acid sequences of maize, rice and Arabidopsis IDD genes, as well as PCP1 from potato. Alignment of the ID domain. Green shaded areas represent amino acid identity in 70% or more of the sequences. The position of each zinc finger is by a bar and cysteine (C) and histidine (H) residues boxed; filled triangle indicate conserved C residues and open triangle indicate conserved H residues. (For the sake of simplicity, we have renamed the zinc finger order (Z1, Z2, Z3 and Z4); in a previous study (Kozaki et al., 2004) the order was Z1, Z3, Z2 and Z4.) A thick bar at the N-terminal region of the ID-domain shows the putative NLS sequence. The pink triangle represents the position intron unique to ID1 and SbID.

Figure 3

Alignment of C-terminal amino acid sequences encoded by maize, rice and Arabidopsis IDD genes, SbID from sorghum and PCP1 from potato. The C-terminal regions of IDD peptides that contain either the MSATALLQKAA or TR/LDFLG conserved domains, or both, shaded in gray. The dotted line represents the non-conserved amino acid sequence separating the two conserved motifs.

Figure 4

Northern blot showing expression of 7 maize IDD genes compared to ID1. Gene-specific probes are indicated on the right side. Tissues used include: S, stem below the apical region, Ap, apical region of plant, including 2 cm above apical meristem and leaf primordia. Sections 1, 2 and 3 are immature leaf sections from 2–4 cm, 4–6 cm and 6–8 cm above the apex, respectively. TL, immature flag leaf surrounding tassel primordium; T, pre-anthesis tassel; R, roots. The same B73 plant with 7 visible leaves was used for sections S, Ap, 1, 2 and 3. (A) Gene-specific probes derived from cDNAs of ZmIDDp1, ZmIDDp10, ZmIDDveg7 and ZmIDDveg9. The ZmCDC2 probe, which is detected in tissues with actively dividing cells, was used as a control [38]. An arrowhead indicates the position of the ZmIDDp10 band. (B) Blot hybridized with specific probes to ZmIDD3, ZmIDD7, ZmIDD13 and an actin-specific probe.

Figure 5

Alignment of entire peptide sequences of ID1, OsID and SbID. Amino acid residues shared by all three peptides are shaded. Each zinc finger is indicated with a light colored box; the putative NLS is shown by a dark bar and the TRDFLG motif is boxed. The boxed area with dotted line shows the C-terminal peptide region used to generate anti-ID1 specific antibody and corresponding sequences in rice and sorghum.

Figure 6

Western blot analysis of ID1, OsID and SbID protein accumulation in maize, rice and sorghum tissues with anti-ID antibody. Ap, apical region containing shoot meristem and leaf primordia; ImL, Immature leaves, approximately 2 to 8 cm above the apical region; ML, mature green leaf blades; FP, floral primordial tissues, which included ear tissue for maize, and yet to emerge spikelet primordia for rice and sorghum. Mature spikelet inflorescence, F, was also examined for rice and sorghum. Arrows at left indicate molecular weight standards of 84 kD and 61 kD.

Figure 7

Phylogenetic comparison of IDD genes from several plant species. Mid-point rooted phylogram (branches proportional to the expected number of substitutions per site) based on genomic DNA sequences, reconstructed using Mr. Bayes. IDD genes found in maize (red), rice (blue) and Arabidopsis (black), as well as PCP1 from potato (Solanum tuberosum; green) and a potential ortholog of ID1 from Sorghum bicolor, SbID (purple), with Bayesian posterior probability values above 80% indicated above the branches.

Figure 8

Phylogenetic comparison of IDD amino acid sequences from several plant species. Mid-point rooted phylogram based on amino acid sequences for the same region used in Figure 7. IDD genes found in maize (red), rice (blue) and Arabidopsis (black), as well as PCP1 from potato (Solanum tuberosum; green) and a potential ortholog of ID1 from Sorghum bicolor, SbID (purple), with Bayesian posterior probability values above 80% indicated above the branches. Circles of the same colour and labeled with the same letter on both trees highlight differences between the two sets of analyses; in particular, circled groups indicate amino acid groupings between Arabidopsis and grasses that were not apparent in genomic DNA analyses. Sequences enclosed by rectangles labeled 'D' form a less-well supported Arabidopsis-grass amino acid assemblage.