Characterization of C‐terminal domains of Arabidopsis heat stress transcription factors (Hsfs) and identification of a new signature combination of plant class A Hsfs with AHA and NES motifs essential for activator function and intracellular localization

Abstract

SummaryHeat stress transcription factors (Hsfs) are the major regulators of the plant heat stress (hs) response. Sequencing of the Arabidopsis genome revealed the existence of 21 open-reading frames (ORFs) encoding putative Hsfs assigned to classes A±C. Here we present results of a functional genomics approach to the Arabidopsis Hsf family focused on the analysis of their C-terminal domains (CTDs) harboring conserved modules for their function as transcription factors and their intracellular localization. Using reporter assays in tobacco protoplasts and yeast as well as glutathione-S-transferase (GST) pull-down assays, we demonstrate that short peptide motifs enriched with aromatic and large hydrophobic amino acid (aa) residues embedded in an acidic surrounding (AHA motifs) are essential for transcriptional activity of class A Hsfs. In contrast to this, class B and C Hsfs lack AHA motifs and have no activator function on their own. We also provide evidence for the function of a leucine (Leu)-rich region centered around a conserved QMGFL motif at the very C-terminus as a nuclear export signal (NES) of class A Hsfs. Sequence comparison indicates that the combination of a C-terminal AHA motif with the consensus sequence FWxxF/L,F/I/L as well as the adjacent NES represents a signature domain for plant class A Hsfs, which allowed to identify more than 60 new Hsfs from the expressed sequence tag (EST) database.

Role of Heat Stress Transcription Factor HsfA5 as Specific Repressor of HsfA4

“…All Arabidopsis Hsf fusion constructs used in this study were described earlier (14) and were kindly provided by Pascal von KoskullDöring (Goethe-University, Frankfurt, Germany). The strength of interaction was confirmed by colony growth in the presence of the histidine biosynthesis inhibitor 3-aminotriazole.…”

“…Detailed investigations on the structural and functional diversification so far are restricted to Hsfs A1, A2, A3, A9, and B1 (10,11,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). 3 An interesting new aspect of functional diversification of Hsfs was reported when a mutant of rice (spl7) with spontaneous necrotic lesions in mature leaves was identified as a point mutation with a Trp Ͼ Cys substitution in the N-terminal part of the DNA binding domain of HsfA4d.…”

“…1A) with an N-terminal DNA binding domain, an adjacent domain with heptad hydrophobic repeats (HR-A/B) involved in oligomerization, a cluster of basic amino acid residues essential for nuclear import (nuclear localization signal), and a C-terminal activation domain, which is frequently characterized by a nuclear export signal and short peptide motifs (AHA motifs) essential for the activator function (10,(12)(13)(14). Three classes of plant Hsfs (classes A, B, and C) are defined by peculiarities of their HR-A/B regions (6,8).…”

The Heat Shock Factor A4A Confers Salt Tolerance and Is Regulated by Oxidative Stress and the Mitogen-Activated Protein Kinases MPK3 and MPK6

“…The N-terminal DNA-binding domain is responsible for the HSE recognition in the promoters of HSF target genes. The hydrophobic heptad repeat region for oligomerization (HRA/B) located close to the DNA-binding domain mediates trimerization, which is prerequisite for their transcription factor activity (Nover et al, 2001;Kotak et al, 2004;Anckar and Sistonen, 2011;Scharf et al, 2012). Accumulating evidence suggests that activation of plant and mammalian HSFs is regulated by a similar mechanism, but the regulation of plant HSFs is much less understood compared with their mammalian counterparts (Akerfelt et al, 2010;Scharf et al, 2012).…”

“…HSFA4 from tomato (Solanum lycopersicum) was shown to activate heat stress-induced genes and interact with HSFA5-generating inactive heterooligomers. HSFA4 and HSFA5 belong to a separate group within the class A HSFs, sharing similarities in the DNAbinding and oligomerization domain and C-terminal conserved sequence motifs (Baniwal et al, 2004;Kotak et al, 2004;Baniwal et al, 2007). In the rice spotted leaf7 mutant, one base substitution in the DNA-binding domain of HSFA4D resulted in the appearance of necrotic lesions on leaves, suggesting stress hypersensitivity (Yamanouchi et al, 2002).…”