Uncharacterized conserved motifs outside the HD-Zip domain in HD-Zip subfamily I transcription factors; a potential source of functional diversity

Abstract

Background: Plant HD-Zip transcription factors are modular proteins in which a homeodomain is associated to a leucine zipper. Of the four subfamilies in which they are divided, the tested members from subfamily I bind in vitro the same pseudopalindromic sequence CAAT(A/T)ATTG and among them, several exhibit similar expression patterns. However, most experiments in which HD-Zip I proteins were over or ectopically expressed under the control of the constitutive promoter 35S CaMV resulted in transgenic plants with clearly different phenotypes. Aiming to elucidate the structural mechanisms underlying such observation and taking advantage of the increasing information in databases of sequences from diverse plant species, an in silico analysis was performed. In addition, some of the results were also experimentally supported.

Results: A phylogenetic tree of 178 HD-Zip I proteins together with the sequence conservation presented outside the HD-Zip domains allowed the distinction of six groups of proteins. A motif-discovery approach enabled the recognition of an activation domain in the carboxy-terminal regions (CTRs) and some putative regulatory mechanisms acting in the amino-terminal regions (NTRs) and CTRs involving sumoylation and phosphorylation. A yeast one-hybrid experiment demonstrated that the activation activity of ATHB1, a member of one of the groups, is located in its CTR. Chimerical constructs were performed combining the HD-Zip domain of one member with the CTR of another and transgenic plants were obtained with these constructs. The phenotype of the chimerical transgenic plants was similar to the observed in transgenic plants bearing the CTR of the donor protein, revealing the importance of this module inside the whole protein.

Conclusions: The bioinformatical results and the experiments conducted in yeast and transgenic plants strongly suggest that the previously poorly analyzed NTRs and CTRs of HD-Zip I proteins play an important role in their function, hence potentially constituting a major source of functional diversity among members of this subfamily.

Figure 1

Phylogenetic trees of HD-Zip I transcription factors. Maximum Likelihood phylogenetic trees were constructed using the amino acid sequences of 178 HD-Zip subfamily I transcription factors from different plant species including monocots, dicots, mosses, ferns and conifers. The HZT was constructed with the sequences of the HD and HALZ domains and is the reference tree. The CST was calculated with the complete sequences. Clades highlighted with different colours represent groups of transcription factors sharing common motifs in their CTRs. These clades are numbered from I to VI whereas group I is divided in three subgroups named Ia, Ib and Ic. Inside these groups, clades exclusively formed by monocots or dicots transcription factors were labelled with an M or a D, respectively; and their structure was collapsed to ease visualization. Proteins shared between groups in the HZT and CST have been erased from the CST. Unshared members have been marked with an asterisk in the HZT. The group labelled Pp includes all the proteins from the moss Physcomitrella patens. Bootstrap support values, as percentages, are indicated in the nodes. Branches with low bootstrap values (below 50%) have been collapsed, with the exception of the basal branches of groups Ic, III and IV in the HZT which have further support from bootstrap values in the CST (see Table 1) and conserved motifs in the NTRs and CTRs (Figures 2, 4 and Additional file 8).

Figure 2

Sequence logos of CTRs from the six groups identified. The sequence logos were constructed with the alignment of the CTRs of the proteins belonging to each of the six groups, including subgroups Ia, Ib and Ic. The height of the residues correlates with their frequency in the alignment, which allows the recognition of clearly conserved regions.

Figure 3

Frequencies of tryptophans in the CTRs. The histogram represents the frequencies of Trp within the CTRs of the 178 proteins according to their relative position in this region, which was divided in ten parts. The last tenth shows a visible enrichment.

Figure 4

Motif location in the CTRs. The 20 motifs found by the program MEME are depicted according to their location in each CTR. The identity of each motif is colour coded according to the legend. Groups are highlighted with a box of dashed boundaries and the phylogenetic relations between the proteins are indicated by the tree on the left side of the plots. Putative phosphorylation sites (Ser, Thr, Tyr) are marked with a black diamond and sumoylation motifs with a blue inverted triangle.

Figure 5

Sequence logos of the motifs found in the CTRs. The sequence logos of the 20 motifs found in the CTRs are sorted according to their relative position. To reflect chemical properties in the distal region, the motifs present in the same row are also combined in many CTRs (except for motifs 9, 10, 19 and 20; some alternative combinations to those shown also exist).

Figure 6

ATHB1 CTR acts as an activation domain in yeast cells. (A) The complete sequence of ATHB1, a version without the CTR (ATHB1WCT), and the CTR alone (ATH1CT) were fused to the DNA-binding domain of GAL4 (GAL4-BD). The empty vector expressing only the GAL4-BD was used as negative control. (B) A β-galactosidase activity assay. (C) Confirming this results, only the CTR and the complete ATHB1 protein had the transactivation activity required to reverse the auxotrophy to His of the AH109 yeast cells, allowing them to grow in medium lacking this amino acid.

Figure 7

Triple response to ethylene in chimerical transgenic plants. (A) Schematic representation of the different constructs used to transform Arabidopsis plants (B) Relative expression levels of the different transgenes in independent lines measured by qPCR. The line with the lowest expression was assigned an unitary level (1). (C) The sensitivity to ethylene was measured analyzing whether the seedlings developed apical hook (sensitive) or not (insensitive). The image exemplifies the phenotypes observed. (D) The results for three different lines from each genotype are presented in the boxplot.

Figure 8

Leaf serration phenotype. Boxplot illustrating the serration grade of the leaves belonging to transgenic plants transformed with different constructs. The images are examples of the analysis conducted with the leaves (HAHB1 on the right and HAHB4 on the left).

Figure 9

Proposed model of functional domains in HD-Zip I transcription factors. The in silico analysis conducted on a considerably large dataset of HD-Zip I transcription factors allowed us to postulate a generalized functional model of this family of proteins. This is supported to some extent by previous studies and the experimental results presented in this work. The well characterized HD-Zip domains are in charge of DNA binding and dimerization, an AHA motif in the CTR is responsible for activation, and the NTR and CTR are regions potentially phosphorylated and sumoylated, depending on the group, thus playing a regulatory role.