Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need?

Sequencing of the Arabidopsis genome revealed a unique complexity of the plant heat stress transcription factor (Hsf) family. By structural characteristics and phylogenetic comparison, the 21 representatives are assigned to 3 classes and 14 groups. Particularly striking is the finding of a new class of Hsfs (AtHsfC1) closely related to Hsf1 from rice and to Hsfs identified from frequently found expressed sequence tags of tomato, potato, barley, and soybean. Evidently, this new type of Hsf is well expressed in different plant tissues. Besides the DNA binding and oligomerization domains (HR-A/B region), we identified other functional modules of Arabidopsis Hsfs by sequence comparison with the well-characterized tomato Hsfs. These are putative motifs for nuclear import and export and transcriptional activation (AHA motifs). There is intriguing flexibility of size and sequence in certain parts of the otherwise strongly conserved N-terminal half of these Hsfs. We have speculated about possible exon-intron borders in this region in the ancient precursor gene of plant Hsfs, similar to the exon-intron structure of the present mammalian Hsf-encoding genes.

The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing alpha-crystallin domains (Acd proteins).

Comprehensive analysis of the Arabidopsis genome revealed a total of 13 sHsps belonging to 6 classes defined on the basis of their intracellular localization and sequence relatedness plus 6 ORFs encoding proteins distantly related to the cytosolic class Cl or the plastidial class of sHsps. The complexity of the Arabidopsis sHsp family far exceeds that in any other organism investigated to date. Furthermore, we have identified a new family of ORFs encoding multidomain proteins that contain one or more regions with homology to the ACD (Acd proteins). The functions of the Acd proteins and the role of their ACDs remain to be investigated.

Plants contain a novel multi-member class of heat shock factors without transcriptional activator potential.

Based on phylogeny of DNA-binding domains and the organization of hydrophobic repeats, two families of heat shock transcription factors (HSFs) exist in plants. Class A HSFs are involved in the activation of the heat shock response, but the role of class B HSFs is not clear. When transcriptional activities of full-length HSFs were monitored in tobacco protoplasts, no class B HSFs from soybean or Arabidopsis showed activity under control or heat stress conditions. Additional assays confirmed the finding that the class B HSFs lacked the capacity to activate transcription. Fusion of a heterologous activation domain from human HSF1 (AD2) to the C-terminus of GmHSFB1-34 gave no evidence of synergistic enhancement of AD2 activity, which would be expected if weak activation domains were present. Furthermore, activity of AtHSFB1-4 (class B) was not rescued by coexpression with AtHSFA4-21 (class A) indicating that the class A HSF was not able to provide a missing function required for class B activity. The transcriptional activation potential of Arabidopsis AtHSFA4-21 was mapped primarily to a 39 amino acid fragment in the C-terminus enriched in bulky hydrophobic and acidic residues. Deletion mutagenesis of the C-terminal activator regions of tomato and Arabidopsis HSFs indicated that these plant HSFs lack heat-inducible regulatory regions analogous to those of mammalian HSF1. These findings suggest that heat shock regulation in plants may differ from metazoans by partitioning negative and positive functional domains onto separate HSF proteins. Class A HSFs are primarily responsible for stress-inducible activation of heat shock genes whereas some of the inert class B HSFs may be specialized for repression, or down-regulation, of the heat shock response.

Isolation and characterization of HsfA3, a new heat stress transcription factor of Lycopersicon peruvianum.

Stress-induced transcription of heat shock proteins (Hsps) in eukaryotes is mediated by a conserved class of transcription factors called heat stress transcription factors (Hsfs). Here we report the isolation and functional characterization of HsfA3, a new member of the Hsf family. HsfA3 was cloned from a tomato heat stress cDNA library by yeast two-hybrid screening, using HsfA1 as a bait. HsfA3 is a single-copy gene with all the conserved sequence elements characteristic of a heat stress transcription factor. The constitutively expressed HsfA3 is mainly found in the cytoplasm under control conditions and in the nucleus under heat stress conditions. Functionally, HsfA3 behaves similarly to the already known members of tomato Hsf family. It is able to substitute yeast Hsf for viability functions and is a strong activator of Hsf-dependent reporter constructs both in tobacco protoplasts and yeast. Finally, similar to the AHA motifs in HsfA1 and HsfA2, the activator function depends on four short peptide motifs with a central tryptophan residue found in the C-terminal domain of HsfA3.

The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules.

In heat-stressed (HS) tomato (Lycopersicon peruvianum) cell cultures, the constitutively expressed HS transcription factor HsfA1 is complemented by two HS-inducible forms, HsfA2 and HsfB1. Because of its stability, HsfA2 accumulates to fairly high levels in the course of a prolonged HS and recovery regimen. Using immunofluorescence and cell fractionation experiments, we identified three states of HsfA2: (i) a soluble, cytoplasmic form in preinduced cultures maintained at 25 degrees C, (ii) a salt-resistant, nuclear form found in HS cells, and (iii) a stored form of HsfA2 in cytoplasmic HS granules. The efficient nuclear transport of HsfA2 evidently requires interaction with HsfA1. When expressed in tobacco protoplasts by use of a transient-expression system, HsfA2 is mainly retained in the cytoplasm unless it is coexpressed with HsfA1. The essential parts for the interaction and nuclear cotransport of the two Hsfs are the homologous oligomerization domain (HR-A/B region of the A-type Hsfs) and functional nuclear localization signal motifs of both partners. Direct physical interaction of the two Hsfs with formation of relatively stabile hetero-oligomers was shown by a two-hybrid test in Saccharomyces cerevisiae as well as by coimmunoprecipitation using tomato and tobacco whole-cell lysates.

Heat stress transcription factors from tomato can functionally replace HSF1 in the yeast Saccharomyces cerevisiae.

The fact that yeast HSF1 is essential for survival under nonstress conditions can be used to test heterologous Hsfs for the ability to substitute for the endogenous protein. Our results demonstrate that like Hsf of Drosophila, tomato Hsfs A1 and A2 can functionally replace the corresponding yeast protein, but Hsf B1 cannot. In addition to survival at 28 degrees C, we checked the transformed yeast strains for temperature sensitivity of growth, induced thermotolerance and activator function using two different lacZ reporter constructs. Tests with full-length Hsfs were supplemented by assays using mutant Hsfs lacking parts of their C-terminal activator region or oligomerization domain, or containing amino acid substitutions in the DNA-binding domain. Remarkably, results with the yeast system are basically similar to those obtained by the analysis of the same Hsfs as transcriptional activators in a tobacco protoplast assay. Most surprising is the failure of HsfB1 to substitute for the yeast Hsf. The defect can be overcome by addition to HsfB1 of a short C-terminal peptide motif from HsfA2 (34 amino acid residues), which represents a type of minimal activator necessary for interaction with the yeast transcription apparatus. Deletion of the oligomerization domain (HR-A/B) does not interfere with Hsf function for survival or growth at higher temperatures. But monomeric Hsf has a markedly reduced affinity for DNA, as shown by lacZ reporter and band-shift assays.

Intracellular distribution and identification of the nuclear localization signals of two plant heat-stress transcription factors.

Similar to heat-stress transcription factors (HSFs) from non-plant sources, HSFA1 and HSFA2 from tomato (Lycopersicon esculentum Mill) contain two conserved clusters of basic amino acid residues (K/R1 and K/R2) which might serve as nuclear localization signal (NLS) motifs. Mutation of either one of them and functional testing of the corresponding proteins in a transient expression assay using tobacco (Nicotiana plumbaginifolia L:) protoplasts gave the following results. Whereas K/R1, positioned in all HSFs at the C-terminus of the DNA-binding domain, had no influence on nuclear import, the K/R1 mutants were impaired in their interaction with the DNA (band-shift assays). In contrast to this, mutants of the K/R2 motif, found 15-20 amino acid residues C-terminal of the oligomerization domain (HR-A/B region), had wild-type activity in DNA-binding but were defective in nuclear import. Thus, for the related tomato HSFA1 and HSFA2 the K/R2 cluster represents the only NLS motif, and in this function it cannot be replaced by K/R1.

The Hsf world: classification and properties of plant heat stress transcription factors.

Based on the partial or complete sequences of 14 plant heat stress transcription factors (Hsfs) from tomato, soybean, Arabidopsis and maize we propose a general nomenclature with two basic classes, i.e. classes A and B each containing two or more types of Hsfs (HsfA1, HsfA2 etc.). Despite some plant-specific peculiarities, essential functional domains and modules of these proteins are conserved among plants, yeast, Drosophila and vertebrates. A revised terminology of these parts follows recommendations agreed upon among the authors and representatives from other laboratories working in this field (see legend to Fig. 1). Similar to the situation with the small heat shock proteins (sHsps), the complexity of the hsf gene family in plants appears to be higher than in other eukaryotic organisms.

Solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24.

Two-dimensional-NMR and three-dimensional-NMR experiments were performed to determine the solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24. Samples of uniformly 15N-labeled and 15N, 13C-labeled recombinant proteins were used in the investigation. A near-complete assignment of the backbone 1H, 15N, and 13C resonances was obtained by three-dimensional triple-resonance experiments, whereas three-dimensional 15N-TOCSY-heteronuclear-single-quantum-correlation-spectroscopy, HCCH-COSY and HCCH-TOCSY spectra were recorded for side-chain assignments, 885 non-redundant distance constraints from two-dimensional-homonuclear and three-dimensional-15N-edited and 13C-edited NOESY spectra and 40 hydrogen-bond constraints from exchange experiments were used for structure calculations. The resulting three-dimensional structure contains a three-helix bundle and a small four-stranded antiparallel beta-sheet that forms a hydrophobic core. The two C-terminal helices are parts of a highly conserved helix-turn-helix motif that is probably involved in DNA recognition and binding. In contrast to heat-stress factors from yeast and animals, the plant heat-stress factors lack a loop of 11 amino acid residues inserted between beta3 and beta4. This leads to a tight turn between these beta-strands.

Functional immobilization of a DNA-binding protein at a membrane interface via histidine tag and synthetic chelator lipids.

The coupling of a DNA-binding protein to self-organized lipid monolayers is examined at the air-water interface by means of film balance techniques and epifluorescence microscopy. We used two recombinant species of the heat shock factor HSF24 which differ only in a carboxy-terminal histidine tag that interacts specifically with the nickel-chelating head group of a synthetic chelator lipid. As key function, HSF24 binds to DNA that contains heat-shock responsible promoter elements. In solution, DNA-protein complex formation is demonstrated for the wild type and fusion protein. Substantial questions of these studies are whether protein function is affected after adsorption to lipid layers and whether a specific docking via histidine tag to the chelator lipid leads to functional immobilization. Using lipid mixtures that allow a lateral organization of chelator lipids within the lipid film, specific binding and unspecific adsorption can be distinguished by pattern formation of DNA-protein complexes. At the lipid interface, functional DNA-protein complexes are only detected, when the histidine-tagged protein was immobilized specifically to a chelator lipid containing monolayer. These results demonstrate that the immobilization of histidine-tagged biomolecules to membranes via chelator lipids is a promising approach to achieve a highly defined deposition of these molecules at an interface maintaining their function.

Promoter specificity and deletion analysis of three heat stress transcription factors of tomato.

Transient expression assays in transformed tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts were used to test the activity of three tomato heat stress transcription factors, HSF24, HSF8 and HSF30, in a trans-activation and a trans-repression assay. The results document differences between the three HSFs with respect to their response to the configuration of heat stress promoter elements (HSEs) in the reporter construct (promoter specificity) and to the stress regime used for activation. Analysis of C-terminal deletions identified acidic sequence elements with a central tryptophan residue, which are important for HSF activity control. Surprisingly, heterologous HSFs from Drosophila and human cells, but not from yeast, were also functional as heat stress-induced transcription factors in this tobacco protoplast system.

Three tomato genes code for heat stress transcription factors with a region of remarkable homology to the DNA-binding domain of the yeast HSF.

Heat stress (hs) treatment of cell cultures of Lycopersicon peruvianum (Lp, tomato) results in activation of preformed transcription factor(s) (HSF) binding to the heat stress consensus element (HSE). Using appropriate synthetic HSE oligonucleotides, three types of clones with potential HSE binding domains were isolated from a tomato lambda gt11 expression library by DNA-ligand screening. One of the potential HSF genes is constitutively expressed, the other two are hs-induced. Sequence comparison defines a single domain of approximately 90 amino acid residues common to all three genes and to the HSE--binding domain of the yeast HSF. The domain is flanked by proline residues and characterized by two long overlapping repeats. We speculate that the derived consensus sequence is also representative for other eukaryotic HSF and that the existence of several different HSF is not unique to plants.

Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs.

In heat-shocked tomato cell cultures, cytoplasmic heat shock granules (HSGs) are tightly associated with a specific subset of mRNAs coding mainly for the untranslated control proteins. This messenger ribonucleoprotein complex was banded in a CsCl gradient after fixation with formaldehyde (approximately 1.30 g/cm3). It contains all the heat shock proteins and most of the RNA applied to the gradient. During heat shock, a reversible aggregation of HSGs from 15S precursor particles can be shown. These pre-HSGs are not identical to the 19S plant prosomes. Ultrastructural analysis supports the ribonucleoprotein nature of HSGs and their composition of approximately 10-nm precursor particles. A model summarizes our results. It gives a reasonable explanation for the striking conservation of untranslated mRNAs during heat shock and may apply also to animal cells.

Control of ribosome biosynthesis in plant cell cultures under heat-shock conditions. Ribosomal RNA.

The immediate block of ribosome biosynthesis in heat-shocked tomato cell cultures is primarily caused by the complete inhibition of pre-rRNP processing. Depending on the heat-shock conditions synthesis of pre-rRNP goes on, though at a reduced level. Synthesis and/or preservation of pre-rRNP during heat shock as well as its efficient processing in the recovery period are thoroughly improved by preconditioning of cells to the hyperthermic treatment. Such preinduced cultures are characterized by their content of preformed heat-shock proteins, whose dominant representative (hsp 70) becomes highly enriched in the characteristic granular rRNP material observed in nucleoli of heat-shocked cells. This is shown by immune fluorescence staining and microautoradiography.

Heat shock induced changes of plant cell ultrastructure and autoradiographic localization of heat shock proteins.

Treating tomato cell cultures and leaves by a physiological heat shock (hs) at 35 to 39 degrees C results in a progressive disintegration of the nucleolus and the assembly of cytoplasmic hs granules. Other ultrastructural changes are not observed. The alterations of the nucleoli coincide with an immediate stop of the processing and with a strongly decreased synthesis of pre-rRNA. Both hs effects are reversed after shift-down to normal temperature conditions (25 degrees C). Assembly of cytoplasmic hs granules depends on the accumulation of the newly forming hs proteins and on supraoptimal temperatures. It is not observed in preinduced cultures synthesizing hs proteins at 25 degrees C. Autoradiographic studies reveal the preferential accumulation of hsp in the nucleoli and hs granules. Furthermore uridine labeling points to the presence of RNA in electron dense particles of both subcellular components. A survey on the state of hsp synthesis and structural binding as well as on the ultrastructural changes is given for 12 selected hs regimes.

Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures.

Synthesis of about 30 acidic and 18 basic heat-shock proteins (hsps) is induced in suspension cultures of tomato (Lycopersicon peruvianum) if subjected to supraoptimal temperature conditions (35-40 degrees C). A characteristic aspect of the plant heat-shock response is the formation of cytoplasmic granular aggregates, heat-shock granules, containing distinct heat-shock proteins as major structural components and, in addition, several hitherto undetected minor acidic and basic heat-shock proteins. Structural binding of heat-shock proteins, i.e. assembly of heat-shock granules, is dependent on the persistance of supraoptimal temperature conditions. Despite the ongoing synthesis also at 25 degrees C, e.g. in pulse heat-shocked cultures, these proteins are accumulated exclusively in soluble form. Individual heat-shock proteins are characterized by their kinetics of synthesis and are classified by their compartmentation behaviour into class A proteins (exclusively found in soluble form, e.g. hsps 95 and 80), class B proteins (5-10% bound to heat-shock granules, e.g. hsps 70, 68), class C proteins (30-80% bound to heat-shock granules, e.g. hsps 21, 17, 15) and class D proteins, which are minor heat-shock proteins only detected in structure-bound form. Major representatives are modified proteins, i.e. hsps 95, 80, 70 and 68 are phosphorylated and hsps 80, 74, 70 and 17 are methylated proteins (numbers 70, 80 etc. refer to 10(-3) Mr). Under heat-shock conditions synthesis of the proteins detected in control cells (25 degrees C proteins) exhibits two patterns. There are proteins with continued and proteins with discontinued synthesis. Synthesis of most of the latter proteins is resumed very rapidly after shift-down to 25 degrees C, even in the presence of actinomycin D. We conclude that reversible segregation of distinct mRNA species from the translation apparatus contributes to the heat-shock-specific pattern of protein synthesis in plants also.