Klaus-Dieter Scharf

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

Abstract 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.

Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factors

Compared to the overall multiplicity of more than 20 plant Hsfs, detailed analyses are mainly restricted to tomato and Arabidopsis and to three important representatives of the family (Hsfs A1, A2 and B1). The three Hsfs represent examples of striking functional diversification specialized for the three phases of the heat stress (hs) response (triggering, maintenance and recovery). This is best illustrated for the tomato Hsf system: (i) HsfA1a is the master regulator responsible for hs-induced gene expression including synthesis of HsfA2 and HsfB1. It is indispensible for the development of thermotolerance. (ii) Although functionally equivalent to HsfA1a, HsfA2 is exclusively found after hs induction and represents the dominant Hsf, the “working horse” of the hs response in plants subjected to repeated cycles of hs and recovery in a hot summer period. Tomato HsfA2 is tightly integrated into a network of interacting proteins (HsfA1a, Hsp17-CII, Hsp17-CI) influencing its activity and intracellular distribution. (iii) Because of structural peculiarities, HsfB1 acts as coregulator enhancing the activity of HsfA1a and/or HsfA2. But in addition, it cooperates with yet to be identified other transcription factors in maintaining and/or restoring housekeeping gene expression.

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.

The plant heat stress transcription factor (Hsf) family: Structure, function and evolution

Ten years after the first overview of a complete plant Hsf family was presented for Arabidopsis thaliana by Nover et al. [1], we compiled data for 252 Hsfs from nine plant species (five eudicots and four monocots) with complete or almost complete genome sequences. The new data set provides interesting insights into phylogenetic relationships within the Hsf family in plants and allows the refinement of their classification into distinct groups. Numerous publications over the last decade document the diversification and functional interaction of Hsfs as well as their integration into the complex stress signaling and response networks of plants. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.

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

Abstract 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 CI 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.

Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves.

Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.

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.

Heat stress proteins and transcription factors

Two discoveries triggered the rapid growth of interest in molecular biological studies in the area of the heat stress response: (i) the finding by F. Ritossa [1] in 1962 of a new pattern of gene activity in polytene chromosomes of Drosophila salivary glands, and (ii) the first description of heat stress-inducible proteins (HSPs) by A. Tissieres and his group in 1974 [2]. A number of important books and reviews can be consulted to reconstruct the remarkable development of the field in the following 20 years (see refs 3–57). In view of the complexity of the hs response, with major features conserved between bacteria, plants, insects and vertebrates, and the central role of members of the HSP families in a constantly increasing number of cellular activities, it is worth recalling the historical roots of experimental work in this field going back to the middle of the last century [37]: In 1864, Julius Sachs [58] reported on an extended series of experiments defining the upper temperature limits of plant growth using a specially designed heat stress chamber for whole plants. The broad interest of plant physiologists in this topic has continued up to the present time and has provided insights into developmental, hormonal, circadian and seasonal influences on the intrinsic and inducible heat resistance of plants [59–65]. Another important root of hs research goes back to the first publication in 1866 by the German physician W. Busch [66] on the spontaneous regression of a skin tumour after local infection with Streptococcus erysipelatis. Following this, W. B. Coley [67] reported in 1893 on 47 cases of treatment of malignant surface tumours by Streptococcus infections or by injection of bacterial extracts (Coley’s toxin). The curative effect is evidently due to hyperthermic damage to the tumour tissue and a local stimulation of the immune system. Hyperthermic treatment of cancer as well as investigations on the basis of cell death under heat stress conditions and survival of tumour cells due to induced thermotolerance became a major part of research in this field and was particularly stimulated after the discovery of induced HSP synthesis [68–72]. Heat stress-induced developmental defects were first described by F. M. Alsop in 1919 [73]. But it was Richard Goldschmidt who elaborated the basis for a developmental genetics in his report in 1935 [74] on the hs-induction of phenocopies of Drosophila developmental mutants. This enormous work was based on the analysis of about 500,000 individuals. His experimental techniques were later extended by N. Petersen and H. K. Mitchell [75] to Drosophila, and J. German [76], summarizing numerous observations in vertebrates, put forward a hypothesis of embryonic stress resulting in formation of abnormal organ anlagen. The brief outline of the early experimental results may help to understand the remarkable velocity and broad scope of scientific development initiated by the discovery of hs-inducible genes and the corresponding proteins [1, 2]. HSPs and the transcription factors regulating their expression (HSFs) will be the focus of this review.

The Tomato Hsf System: HsfA2 Needs Interaction with HsfA1 for Efficient Nuclear Import and May Be Localized in Cytoplasmic Heat Stress Granules

ABSTRACT 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°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 inSaccharomyces cerevisiae as well as by coimmunoprecipitation using tomato and tobacco whole-cell lysates.

The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties

The small heat shock proteins (sHsps), which are ubiquitous stress proteins proposed to act as chaperones, are encoded by an unusually complex gene family in plants. Plant sHsps are classified into different subfamilies according to amino acid sequence similarity and localization to distinct subcellular compartments. In the whole Arabidopsis thaliana genome, 19 genes were annotated to encode sHsps, of which 14 belong to previously defined plant sHsp families. In this paper, we report studies of the five additional sHsp genes in A. thaliana, which can now be shown to represent evolutionarily distinct sHsp subfamilies also found in other plant species. While two of these five sHsps show expression patterns typical of the other 14 genes, three have unusual tissue specific and developmental profiles and do not respond to heat induction. Analysis of intracellular targeting indicates that one sHsp represents a new class of mitochondrion-targeted sHsps, while the others are cytosolic/nuclear, some of which may cooperate with other sHsps in formation of heat stress granules. Three of the five new proteins were purified and tested for chaperone activity in vitro. Altogether, these studies complete our basic understanding of the sHsp chaperone family in plants.