Characterization of the class IV homeodomain-Leucine Zipper gene family in Arabidopsis

Abstract

The Arabidopsis (Arabidopsis thaliana) genome contains 16 genes belonging to the class IV homeodomain-Leucine zipper gene family. These include GLABRA2, ANTHOCYANINLESS2, FWA, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), and PROTODERMAL FACTOR2 (PDF2). Our previous study revealed that atml1 pdf2 double mutants have severe defects in the shoot epidermal cell differentiation. Here, we have characterized additional members of this gene family, which we designated HOMEODOMAIN GLABROUS1 (HDG1) through HDG12. Analyses of transgenic Arabidopsis plants carrying the gene-specific promoter fused to the bacterial beta-glucuronidase reporter gene revealed that some of the promoters have high activities in the epidermal layer of the shoot apical meristem and developing shoot organs, while others are temporarily active during reproductive organ development. Expression profiles of highly conserved paralogous gene pairs within the family were found to be not necessarily overlapping. Analyses of T-DNA insertion mutants of these HDG genes revealed that all mutants except hdg11 alleles exhibit no abnormal phenotypes. hdg11 mutants show excess branching of the trichome. This phenotype is enhanced in hdg11 hdg12 double mutants. Double mutants were constructed for other paralogous gene pairs and genes within the same subfamily. However, novel phenotypes were observed only for hdg3 atml1 and hdg3 pdf2 mutants that both exhibited defects in cotyledon development. These observations suggest that some of the class IV homeodomain-Leucine zipper members act redundantly with other members of the family during various aspects of cell differentiation. DNA-binding sites were determined for two of the family members using polymerase chain reaction-assisted DNA selection from random oligonucleotides with their recombinant proteins. The binding sites were found to be similar to those previously identified for ATML1 and PDF2, which correspond to the pseudopalindromic sequence 5'-GCATTAAATGC-3' as the preferential binding site.

Figure 1.

Identity of the Arabidopsis HD-ZIP IV genes. A, Phylogenetic tree showing the predicted relationships between all members of the class III and class IV HD-ZIP proteins of Arabidopsis. Full-length amino acid sequences of each protein were aligned using CLUSTAL W (Thompson et al., 1994) and revised manually. The tree was constructed by using the neighbor-joining method (Saitou and Nei, 1987). Paralogous gene pairs that likely arose via large-scale genome duplications are shaded. B, Sequence comparisons of the HD helix-III region between different classes of the Arabidopsis HD proteins. Two representative members of each family are aligned with all members of HD-ZIP III and IV families. Residues identical to those of GL2 are indicated by dashes. Asterisks denote potential DNA sequence-specific contact residues (Gehring et al., 1994). C, Sequence comparison of the HD helix-III region, followed by the zipper-loop-zipper motif, between the HD-ZIP IV proteins from different plant species. Zm, Pa, and Moss indicate maize, Picea abies, and P. patens, respectively. Residues identical to those of the Arabidopsis HDG1 are indicated by dashes. Conserved Cyss implicated in redox regulation (Tron et al., 2002) are shaded. GenBank accession numbers are Y17898 (ZmOCL1) and AF172931 (PaHB1). Moss ZIP IV is ASYB17005 in the Physcomitrella EST database (http://moss.nibb.ac.jp/).

Figure 2.

Genomic structures of the Arabidopsis HD-ZIP IV genes and locations of T-DNA insertions. Exons are indicated by boxes, whereas introns are indicated by lines. Black and gray areas represent the regions coding for the HD and the START domain, respectively. Arrowheads indicate the location of the T-DNA insertions. Arrows indicate putative transcription start sites, which are based on the full-length cDNA sequences deposited in GenBank.

Figure 3.

Determination of the consensus binding sequences for the HD-ZIP IV proteins using PCR-assisted random oligonucleotide selection. The sequences selected by MBP fusion proteins with ML1, HDG7, HDG9, and PDF2 were aligned and tabulated. The number of occurrences of the four bases and the derived consensus sequences are shown. n, The total number of oligonucleotides that are derived from random sequence positions.

Figure 4.

RT-PCR analysis of the HDG transcript levels in different organs. Total RNA was extracted from 7-d-old seedlings (Sd), roots of 14-d-old seedlings grown on Murashige and Skoog agar medium (Rt), leaves (Lf), stems (St), flowers (Fl), and siliques (Sl). The latter four organ samples were prepared from 40-d-old flowering plants. Values below the gel represent the ratio of each transcript to ACT8, with the maximum value set to 100.

Figure 5.

GUS expression patterns in the seedlings of HDG promoter-GUS transgenic lines. A, HDG1-GUS expression in developing trichomes. B to H, HDG2-GUS expression in the root hairless cell file of hypocotyls (B and C), developing leaves (D), trichomes (E), shoot apical meristems, leaf primordia, stipules (F and G), and stomatal meristemoids (H). I to M, HDG5-GUS expression in developing leaves (I), trichomes (J), shoot apical meristems, leaf primordia, stipules (K and L), and stomatal meristemoids (M). N to P, HDG7-GUS expression at the base of leaf primordia. Q to T, HDG11-GUS expression in cotyledon margins and developing leaves (Q), trichomes (R), shoot apical meristems, leaf primordia, and stipules (S and T). U to X, HDG12-GUS expression in developing leaves (U), trichomes (V), shoot apical meristems, leaf primordia, and stipules (W and X). A transverse section through hypocotyls (C) was prepared from a 5-d-old seedling of the HDG2-GUS line. Longitudinal sections (F, K, O, S, and W) and transverse sections (G, L, P, T, and X) through shoot apices were prepared from 5- to 7-d-old seedlings of respective plant lines.

Figure 6.

GUS expression patterns in the root tissue of HDG promoter-GUS transgenic lines. A, HDG1-GUS expression in two cell lines at the edge of emergent lateral roots. B, HDG2-GUS expression in primary root tips. C to E, HDG7-GUS expression in lateral root primordia, shown by a transverse section of primary roots (C), emerging lateral root tips (D), and primary root tips (E). F, HDG11-GUS expression in emerging lateral root tips. G, HDG12-GUS expression in emerging lateral root tips.

Figure 7.

GUS expression patterns in the flowers of HDG promoter-GUS transgenic lines. A and B, HDG1-GUS expression in filaments. C to E, HDG2-GUS expression in inflorescence meristems (C), carpels (D and E), and ovules (E). F to J, HDG5-GUS expression in inflorescence meristems (F), filaments (G), carpels (G–J), and ovules (H and I). K and L, HDG9-GUS expression in anthers. M and N, HDG10-GUS expression in anthers. O to R, HDG11-GUS expression in inflorescence meristems (O), stigma (P and Q), petals (P and R), carpels (P–R), and ovules (Q and R). S to U, HDG12-GUS expression in inflorescence meristems (S), stigma (S and T), filaments (T), carpels (T and U), and ovules (U). Transverse sections (B, I, L, N, and R) and longitudinal sections (E, H, Q, and U) through floral organs were prepared from open flowers. Longitudinal sections through inflorescence apices (C, F, O, and S) were prepared from 30-d-old plants of the respective lines.

Figure 8.

GUS expression patterns during embryo development of HDG promoter-GUS transgenic lines. A to F, HDG2-GUS expression in the chalazal end of the embryo sac (A and B), endosperms (C and D), seed coats (E), and embryos (E and F). G to I, HDG5-GUS expression in endosperms (G and H) and embryos (H and I). J to L, HDG7-GUS expression in epidermal boundaries of two cotyledons of embryos. M and N, HDG8-GUS expression in endosperms and embryos. O, HDG9-GUS expression in the chalazal end of the embryo sac. P and Q, HDG11-GUS expression in embryos. R and S, HDG12-GUS expression in embryos.

Figure 9.

HDG11 and HDG12 act in the regulation of branching of the trichome. Typical examples of the trichome on the third leaf of 15-d-old wild-type (A), hdg11-1 (B), hdg12-2 (C), hdg11-1 hdg12-2 (D), gl2-t1 (E), and hdg11-1 gl2-t1 (F) seedlings are shown. The excess branching of the trichome in hdg11-1 is enhanced in hdg11-1 hdg12-2.

Figure 10.

HDG3 acts in the cotyledon development in cooperation with ATML1 and PDF2. Ten-day-old seedlings of hdg2-3 hdg3-1 (A), atml1-1 hdg3-1 (B), and pdf2-1 hdg3-1 (C–I) are shown. Hyponasty of atml1-1 hdg3-1 cotyledons is indicated by arrows in B. pdf2-1 hdg3-1 mutants exhibit a variety of morphological defects in cotyledons such as altered form (C), nonopposite arrangement (D), cotyledon fusion (E), pin (F), and adventitious shoot formation (G). H and I are a magnified view and a section of the adventitious shoot shown by an arrow in G, respectively.