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    Klaus-Dieter Scharf

    Heat stress transcription factors (HSFs) regulate transcriptional response to a large number of environmental influences, such as temperature fluctuations and chemical compound applications. Plant HSFs represent a large and diverse gene... more
    Heat stress transcription factors (HSFs) regulate transcriptional response to a large number of environmental influences, such as temperature fluctuations and chemical compound applications. Plant HSFs represent a large and diverse gene family. The HSF members vary substantially both in gene expression patterns and molecular functions. HEATSTER is a web resource for mining, annotating, and analyzing members of the different classes of HSFs in plants. A web-interface allows the identification and class assignment of HSFs, intuitive searches in the database and visualization of conserved motifs, and domains to classify novel HSFs.
    Plants code for a multitude of heat stress transcription factors (Hsfs). Three of them act as central regulators of heat stress (HS) response in tomato (Solanum lycopersicum). HsfA1a regulates the initial response, and HsfA2 controls... more
    Plants code for a multitude of heat stress transcription factors (Hsfs). Three of them act as central regulators of heat stress (HS) response in tomato (Solanum lycopersicum). HsfA1a regulates the initial response, and HsfA2 controls acquired thermotolerance. HsfB1 is a transcriptional repressor but can also act as co-activator of HsfA1a. Currently, the mode of action and the relevance of the dual function of HsfB1 remain elusive. We examined this in HsfB1 overexpression or suppression transgenic tomato lines. Proteome analysis revealed that HsfB1 overexpression stimulates the co-activator function of HsfB1 and consequently the accumulation of HS-related proteins under non-stress conditions. Plants with enhanced levels of HsfB1 show aberrant growth and development but enhanced thermotolerance. HsfB1 suppression has no significant effect prior to stress. Upon HS, HsfB1 suppression strongly enhances the induction of heat shock proteins due to the higher activity of other HS-induced Hs...
    Pollen cells possess specialized cellular compartments separated by membranes. Consequently, mature pollen contains proteinaceous factors for inter- and intracellular transport of metabolites or ions to facilitate the upcoming energy... more
    Pollen cells possess specialized cellular compartments separated by membranes. Consequently, mature pollen contains proteinaceous factors for inter- and intracellular transport of metabolites or ions to facilitate the upcoming energy exhausting processes - germination and fertilization. Despite the current advancement in the understanding of pollen development little is known about the role and molecular nature of the membrane proteome that participates in functioning and development of male gametophyte. We dissected the membrane proteome of mature pollen from economically important crop Solanum lycopersicum (tomato). Isolated membrane fractions from mature pollen of two tomato cultivars (cv. Moneymaker and cv. Red setter) were subjected to shotgun proteomics (GEL-LC-Orbitrap-MS). The global tomato protein assignment was achieved by mapping the peptides on reference genome (cv. Heinz 1706) and de novo assembled transcriptome vased on mRNA sequencing from the respective cultivar. We identified 687 proteins, where 176 were assigned as putative membrane proteins. About 58% of the identified membrane proteins participate in transport processes. In depth analysis revealed proteins corresponding to energy related pathways (Glycolysis and Krebs cycle) as prerequisite for mature pollen, thereby revealing a reliable model of energy reservoir of the male gametophyte. Mature pollen plays an indispensable role in plant fertility and crop production. To decipher the functionality of pollen global proteomics studies have been undertaken. However, these datasets are deficient in membrane proteins due to their low abundance and solubility. The work presented here provides a comprehensive investigation of membrane proteome of male gametophyte of an agriculturally important crop plant tomato. The analysis of membrane enriched fractions from two tomato cultivars ensured an effective profiling of the pollen membrane proteome. Particularly proteins of the Krebs cycle or the glycolysis process have been detected and thus a model for the energy dynamics and preparedness of the male gametophyte for the upcoming events- germination and fertilization is provided.
    The unprecedented role of sncRNAs in the regulation of pollen biogenesis on both transcriptional and epigenetic levels has been experimentally proven. However, little is known about their global regulation, especially under stress... more
    The unprecedented role of sncRNAs in the regulation of pollen biogenesis on both transcriptional and epigenetic levels has been experimentally proven. However, little is known about their global regulation, especially under stress conditions. We used tomato pollen in order to identify pollen stage-specific sncRNAs and their target mRNAs. We further deployed elevated temperatures to discern stress responsive sncRNAs. For this purpose high throughput sncRNA-sequencing as well as Massive Analysis of cDNA Ends (MACE) were performed for three-replicated sncRNAs libraries derived from tomato tetrad, post-meiotic, and mature pollen under control and heat stress conditions. Using the omiRas analysis pipeline we identified known and predicted novel miRNAs as well as sncRNAs from other classes, responsive or not to heat. Differential expression analysis revealed that post-meiotic and mature pollen react most strongly by regulation of the expression of coding and non-coding genomic regions in ...
    data base containing sequences and analysis tools for 850 Hsfs from 33 plants with full length sequence available, see:http://www.cibiv.at/services/hsf/ (Scharf, Berberich, Ebersberger, Nover BBA 2012)
    Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by HS... more
    Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by HS transcription factors (Hsfs). The role of Hsfs and Hsps in HS response in tomato was initially examined by transcriptome analysis using the massive analysis of cDNA ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points towards two major chaperone networks. One cluster consisted of constitutively expressed plastidial chap...
    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... more
    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...
    Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 (Hsp70) and 90 (Hsp90) assist the transport of preproteins to organelles such as... more
    Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 (Hsp70) and 90 (Hsp90) assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein (Chip), appears to have a more general role in the control of cytosolic protein ...
    Cell survival under high temperature conditions involves the activation of heat stress response (HSR), which in principle is highly conserved among different organisms, but shows remarkable complexity and unique features in plant systems.... more
    Cell survival under high temperature conditions involves the activation of heat stress response (HSR), which in principle is highly conserved among different organisms, but shows remarkable complexity and unique features in plant systems. The transcriptional reprogramming at higher temperatures is controlled by the activity of the heat stress transcription factors (Hsfs). Hsfs allow the transcriptional activation of HSR genes, among which heat shock proteins (Hsps) are best characterized. Hsps belong to multigene families encoding for molecular chaperones involved in various processes including maintenance of protein homeostasis as a requisite for optimal development and survival under stress conditions. Hsfs form complex networks to activate downstream responses, but are concomitantly subjected to cell-type-dependent feedback regulation through factor-specific physical and functional interactions with chaperones belonging to Hsp90, Hsp70 and small Hsp families. There is increasing ...
    Vesicle transport is a central process to ensure protein and lipid distribution in eukaryotic cells. The current knowledge on the molecular components and mechanisms of this process is majorly based on studies in Saccharomyces cerevisiae... more
    Vesicle transport is a central process to ensure protein and lipid distribution in eukaryotic cells. The current knowledge on the molecular components and mechanisms of this process is majorly based on studies in Saccharomyces cerevisiae and Arabidopsis thaliana, which revealed 240 different proteinaceous factors either experimentally proven or predicted to be involved in vesicle transport. In here, we performed an orthologue search using two different algorithms to identify the components of the secretory pathway in yeast and 14 plant genomes by using the 'core-set' of 240 factors as bait. We identified 4021 orthologues and (co-)orthologues in the discussed plant species accounting for components of COP-II, COP-I, Clathrin Coated Vesicles, Retromers and ESCRTs, Rab GTPases, Tethering factors and SNAREs. In plants, we observed a significantly higher number of (co-)orthologues than yeast, while only 8 tethering factors from yeast seem to be absent in the analyzed plant genome...
    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... more
    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-typ...
    Heat stress transcription factors (Hsfs) regulate gene expression in response to environmental stress. The Hsf network in plants is controlled at the transcriptional level by cooperation of distinct Hsf members and by interaction with... more
    Heat stress transcription factors (Hsfs) regulate gene expression in response to environmental stress. The Hsf network in plants is controlled at the transcriptional level by cooperation of distinct Hsf members and by interaction with chaperones. We found two general mechanisms of Hsf regulation by chaperones while analyzing the three major Hsfs, A1, A2, and B1, in tomato (Solanum lycopersicum). First, Hsp70 and Hsp90 regulate Hsf function by direct interactions. Hsp70 represses the activity of HsfA1, including its DNA binding, and the coactivator function of HsfB1 in the complex with HsfA2, while the DNA binding activity of HsfB1 is stimulated by Hsp90. Second, Hsp90 affects the abundance of HsfA2 and HsfB1 by modulating hsfA2 transcript degradation involved in regulation of the timing of HsfA2 synthesis. By contrast, HsfB1 binding to Hsp90 and to DNA are prerequisites for targeting this Hsf for proteasomal degradation, which also depends on a sequence element in its carboxyl-termi...
    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... more
    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.
    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... more
    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.
    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... more
    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 heat stress response is universal to all organisms. Upon elevated temperatures, heat stress transcription factors (Hsfs) are activated to up-regulate the expression of molecular chaperones to protect cells against heat damages. In... more
    The heat stress response is universal to all organisms. Upon elevated temperatures, heat stress transcription factors (Hsfs) are activated to up-regulate the expression of molecular chaperones to protect cells against heat damages. In higher plants, the phenomenon is unusually complex both at the level of Hsfs and heat stress proteins (Hsps). Over-expression of both Hsfs and Hsps and the use of RNA interference for gene knock-down in a transient system in tomato protoplasts allowed us to dissect the in vivo chaperone functions of essential components of thermotolerance, such as the cytoplasmic sHsp, Hsp70 and Hsp100 chaperone families, and the regulation of their expression. The results point to specific functions of the different components in protection from protein denaturation and in refolding of denatured proteins.
    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... more
    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.
    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... more
    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 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.
    Heat shock of cell suspension cultures of tomato (Lycopersicon peruvianum) results in a rapid and reversible decline of the phosphorylation level of a single basic ribosomal protein of the small subunit (tentatively identified as... more
    Heat shock of cell suspension cultures of tomato (Lycopersicon peruvianum) results in a rapid and reversible decline of the phosphorylation level of a single basic ribosomal protein of the small subunit (tentatively identified as ribosomal protein S6). Simultaneously, phosphate labeling of several acidic ribosomal proteins of the large subunit is enhanced. Data on the temperature-dependent distribution of S6 subspecies and on the kinetics and reversibility of S6 phosphorylation are given. The decreased phosphorylation of S6 at temperatures higher than 35 degrees C coincides with the onset of heat shock protein synthesis and precedes a decline of the mitotic index. Recovery from heat shock is characterized by S6 rephosphorylation and, subsequently, leads to an abnormally high mitotic index.
    Background Protein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and... more
    Background Protein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and chloroplasts have been established based on the analysis of model organisms. Today, these results have to be transferred to other plant species. We bioinformatically determined the inventory of putative translocation factors in tomato (Solanum lycopersicum) by orthologue search and domain architecture analyses. In addition, we investigated the diversity of such systems by comparing our findings to the model organisms Saccharomyces cerevisiae, Arabidopsis thaliana and 12 other plant species. Results The literature search end up in a total of 130 translocation components in yeast and A. thaliana, which are either experimentally confirmed or homologous to experimentally confirmed factors. From our bioinformatic analysis (PGAP and OrthoMCL), we identifie...
    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... more
    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.
    tRNA-NTs (tRNA nucleotidyltransferases) are required for the maturation or repair of tRNAs by ensuring that they have an intact cytidine-cytidine-adenosine sequence at their 3′-termini. Therefore this enzymatic activity is found in all... more
    tRNA-NTs (tRNA nucleotidyltransferases) are required for the maturation or repair of tRNAs by ensuring that they have an intact cytidine-cytidine-adenosine sequence at their 3′-termini. Therefore this enzymatic activity is found in all cellular compartments, namely the nucleus, cytoplasm, plastids and mitochondria, in which tRNA synthesis or translation occurs. A single gene codes for tRNA-NT in plants, suggesting a complex targeting mechanism. Consistent with this, distinct signals have been proposed for plastidic, mitochondrial and nuclear targeting. Our previous research has shown that in addition to N-terminal targeting information, the mature domain of the protein itself modifies targeting to mitochondria and plastids. This suggests the existence of an as yet unknown determinate for the distribution of dual-targeted proteins between these two organelles. In the present study, we explore the enzymatic and physicochemical properties of tRNA-NT variants to correlate the properties...
    Abstract Six heat shock transcription factors (HSFs) have been isolated and characterized from soybean and two from Arabidopsis (Czarnecka-Verner et al. 1995; Barros, Czarnecka-Verner and Gurley, unpublished). Based on a phylogeny... more
    Abstract Six heat shock transcription factors (HSFs) have been isolated and characterized from soybean and two from Arabidopsis (Czarnecka-Verner et al. 1995; Barros, Czarnecka-Verner and Gurley, unpublished). Based on a phylogeny analysis of the DNA binding ...