Plant homeodomain-leucine zipper I transcription factors exhibit different functional AHA motifs that selectively interact with TBP or/and TFIIB

Abstract

Different members of the HD-Zip I family of transcription factors exhibit differential AHA-like activation motifs, able to interact with proteins of the basal transcriptional machinery. Homeodomain-leucine zipper proteins are transcription factors unique to plants, classified in four subfamilies. Subfamily I members have been mainly associated to abiotic stress responses. Several ones have been characterized using knockout or overexpressors plants, indicating that they take part in different signal transduction pathways even when their expression patterns are similar and they bind the same DNA sequence. A bioinformatic analysis has revealed the existence of conserved motifs outside the HD-Zip domain, including transactivation AHA motifs. Here, we demonstrate that these putative activation motifs are functional. Four members of the Arabidopsis family were chosen: AtHB1, AtHB7, AtHB12 and AtHB13. All of them exhibited activation activity in yeast and in plants but with different degrees. The protein segment necessary for such activation was different for these four transcription factors as well as the role of the tryptophans they present. When interaction with components of the basal transcription machinery was tested, AtHB1 was able to interact with TBP, AtHB12 interacted with TFIIB, AtHB7 interacted with both, TBP and TFIIB while AtHB13 showed weak interactions with any of them, in yeast two-hybrid as well as in pull-down assays. Transient transformation of Arabidopsis seedlings confirmed the activation capacity and specificity of these transcription factors and showed some differences with the results obtained in yeast. In conclusion, the differential activation functionality of these transcription factors adds an important level of functional divergence of these proteins, and together with their expression patterns, these differences could explain, at least in part, their functional divergence.

Arabidopsis thaliana HomeoBox 1 (AtHB1), a Homedomain‐Leucine Zipper I (HD‐Zip I) transcription factor, is regulated by PHYTOCHROME‐INTERACTING FACTOR 1 to promote hypocotyl elongation

“…The pCAMBIA‐His vector was constructed by cloning the segment of the pBI122 vector (Capella et al ., ), containing the 35S cauliflower mosaic virus ( CaMV ), the His‐tag, the multiple cloning site and the NOPALINE SYNTHASE into the Hin dIII and Eco RI sites of pCAMBIA1380 .…”

“…To generate 35S::AtHB1, the GST::AtHB1 clone was digested with BamHI to clone it into the unique BamHI site of pBI122 (Capella et al, 2014). The insert orientation was determined by sequencing.…”

“…In a recent phylogenetic reconstruction using 178 HD-Zip I proteins from different species that considered not only the HD-Zip motif but also the carboxy-terminal regions (CTRs), A. thaliana HD-Zip I proteins were classified into six groups, named I-VI (Arce et al, 2011). These CTRs were demonstrated to be responsible for the interaction with other TFs and for transactivation (Capella et al, 2014).…”

“…Arabidopsis thaliana HomeoBox 1 (AtHB1) belongs to group III of HD-Zip subfamily I and acts as a transcriptional activator (Arce et al, 2011;Capella et al, 2014). Point mutations in AtHB1 CTR followed by activation assays in yeast and plants indicated that this TF can interact with Arabidopsis thaliana TATA BINDING PROTEIN 2, a component of the basal transcription machinery (Capella et al, 2014). The expression of this gene is down-regulated by NaCl and low temperatures and up-regulated in darkness (Henriksson et al, 2005).…”

The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress

“…The complete OsWRKY47 cDNA sequence was amplified by PCR with the W47R and W47F primers using the Oswrky47cd clone and as template cloned into the pBI122 vector (Capella et al 2014) in the BamHI and KpnI restriction sites (Table S3). This construct was used to transform tobacco leaves.…”