Lothar Altschmied

Seed-specific transcription factors ABI3 and FUS3: molecular interaction with DNA

In Arabidopsis thaliana (L.) Heynh. the seed-specific transcription factors ABI3 and FUS3 have key regulatory functions during the development of mature seeds. The highly conserved RY motif [DNA motif CATGCA(TG)], present in many seed-specific promoters, is an essential target of both regulators. Here we show that, in vitro, the full-length ABI3 protein, as well as FUS3 protein, is able to bind to RY-DNA and that the B3 domains of both transcription factors are necessary and sufficient for the specific interaction with the RY element. Flanking sequences of the RY motif modulate the binding, but the presence of an RY sequence alone allows the specific interaction of ABI3 and FUS3 with the target in vitro. Transcriptional activity of ABI3 and FUS3, measured by transient promoter activation, requires the B3 DNA-binding domain and an activation domain. In addition to the known N-terminal-located activation domain, a second transcription activation domain was found in the B1 region of ABI3.

A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single‐copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin‐like C‐terminal segment of 20 kDa, a predicted N‐terminal leucine zipper and a potential phosphatase‐binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non‐appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl‐prolyl cis–trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light‐mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.

The plastocyanin binding domain of photosystem I.

The molecular recognition between plastocyanin and photosystem I was studied. Photosystem I and plastocyanin can be cross‐linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant plastocyanins are cross‐linked with lysine residues of a domain near the N‐terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of plastocyanin by site‐directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha‐helices. The interacting N‐terminal helix lines up six lysines on one side which may guide a fast one‐dimensional diffusion of plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two‐step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.

Genetic mapping and BAC assignment of EST-derived SSR markers shows non-uniform distribution of genes in the barley genome

A set of 111,090 barley expressed sequence tags (ESTs) was searched for the presence of microsatellite motifs [simple sequence repeat (SSRs)] and yielded 2,823 non-redundant SSR-containing ESTs (SSR–ESTs). From this, a set of 754 primer pairs was designed of which 525 primer pairs yielded an amplicon and as a result, 185 EST-derived microsatellite loci (EST–SSRs) were placed onto a genetic map of barley. The markers show a uniform distribution along all seven linkage groups ranging from 21 (7H) to 35 (3H) markers. Polymorphism information content values ranged from of 0.24 to 0.78 (average 0.48). To further investigate the physical distribution of the EST–SSRs in the barley genome, a bacterial artificial chromosomes (BAC) library was screened. Out of 129 markers tested, BAC addresses were obtained for 127 EST–SSR markers. Twenty-seven BACs, forming eight contigs, were hit by two or three EST–SSRs each. This unexpectedly high incidence of EST–SSRs physically linked at the sub-megabase level provides additional evidence of an uneven distribution of genes and the segmentation of the barley genome in gene-rich and gene-poor regions.

Differential gene expression during seed germination in barley (Hordeum vulgare L.)

A barley cDNA macroarray comprising 1,440 unique genes was used to analyze the spatial and temporal patterns of gene expression in embryo, scutellum and endosperm tissue during different stages of germination. Among the set of expressed genes, 69 displayed the highest mRNA level in endosperm tissue, 58 were up-regulated in both embryo and scutellum, 11 were specifically expressed in the embryo and 16 in scutellum tissue. Based on Blast X analyses, 70% of the differentially expressed genes could be assigned a putative function. One set of genes, expressed in both embryo and scutellum tissue, included functions in cell division, protein translation, nucleotide metabolism, carbohydrate metabolism and some transporters. The other set of genes expressed in endosperm encodes several metabolic pathways including carbohydrate and amino acid metabolism as well as protease inhibitors and storage proteins. As shown for a storage protein and a trypsin inhibitor, the endosperm of the germinating barley grain contains a considerable amount of residual mRNA which was produced during seed development and which is degraded during early stages of germination. Based on similar expression patterns in the endosperm tissue, we identified 29 genes which may undergo the same degradation process.

Toward the identification and regulation of the Arabidopsis thaliana ABI3 regulon.

The plant-specific, B3 domain-containing transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) is an essential component of the regulatory network controlling the development and maturation of the Arabidopsis thaliana seed. Genome-wide chromatin immunoprecipitation (ChIP-chip), transcriptome analysis, quantitative reverse transcriptase–polymerase chain reaction and a transient promoter activation assay have been combined to identify a set of 98 ABI3 target genes. Most of these presumptive ABI3 targets require the presence of abscisic acid for their activation and are specifically expressed during seed maturation. ABI3 target promoters are enriched for G-box-like and RY-like elements. The general occurrence of these cis motifs in non-ABI3 target promoters suggests the existence of as yet unidentified regulatory signals, some of which may be associated with epigenetic control. Several members of the ABI3 regulon are also regulated by other transcription factors, including the seed-specific, B3 domain-containing FUS3 and LEC2. The data strengthen and extend the notion that ABI3 is essential for the protection of embryonic structures from desiccation and raise pertinent questions regarding the specificity of promoter recognition.

Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis thaliana

The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467 selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time course from 30 min to 24 h. 97 and 64 genes were identified that were up- or down-regulated more than 2–fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfur-related genes were by far strongest affected. Gene expression and metabolite patterns of sulfur metabolism were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response. These results document for the first time how comprehensively the regulation of sulfur-related genes and plant defense are connected. This interaction is discussed as a new approach to differentiate between supply- and demand-driven regulation of the sulfate assimilation pathway.

Elongation‐related functions of LEAFY COTYLEDON1 during the development of Arabidopsis thaliana

The transcription factor LEAFY COTYLEDON1 (LEC1) controls aspects of early embryogenesis and seed maturation in Arabidopsis thaliana. To identify components of the LEC1 regulon, transgenic plants were derived in which LEC1 expression was inducible by dexamethasone treatment. The cotyledon-like leaves and swollen root tips developed by these plants contained seed-storage compounds and resemble the phenotypes produced by increased auxin levels. In agreement with this, LEC1 was found to mediate up-regulation of the auxin synthesis gene YUCCA10. Auxin accumulated primarily in the elongation zone at the root-hypocotyl junction (collet). This accumulation correlates with hypocotyl growth, which is either inhibited in LEC1-induced embryonic seedlings or stimulated in the LEC1-induced long-hypocotyl phenotype, therefore resembling etiolated seedlings. Chromatin immunoprecipitation analysis revealed a number of phytohormone- and elongation-related genes among the putative LEC1 target genes. LEC1 appears to be an integrator of various regulatory events, involving the transcription factor itself as well as light and hormone signalling, especially during somatic and early zygotic embryogenesis. Furthermore, the data suggest non-embryonic functions for LEC1 during post-germinative etiolation.

Clp protease complexes and their diversity in chloroplasts

Abstract. The Clp proteases represent a large, ancient ATP-dependent protease family which in higher plants is known to be located in chloroplasts. The soluble, presumably multisubunit, enzyme of the organelle stroma is of dual genetic origin. It consists of a nuclear-encoded, regulatory subunit ClpC, which is an ATPase, and a plastid-encoded proteolytic subunit ClpP, which is a serine protease. An additional, nuclear-encoded proteolytic subunit resembling ClpP has been recently reported from tomato (Schaller and Ryan, 1995 plant gene Register 95–00). We demonstrate that in both tomato Lycopersicon esculentum Mill. and Arabidopsis thaliana, (L.) Heynh. the nuclear-encoded ClpP (nClpP) is made as a precursor molecule that can be imported into isolated intact chloroplasts of spinach (Spinacia oleracea L.) and processed in two or three steps, respectively, to the size of the authentic protein. Furthermore, both gel electrophoresis under non-denaturing conditions and size-exclusion chromatography verified that the three proteins can form distinct heteromeric supramolecular complexes of approximately 860, 1380 and 1700 kDa (probably also of 600 kDa) molecular mass. The size ranges of the former two are reminiscent of those of Clp complexes described from Escherichia coli. In addition, various complexes between 160 and 560 kDa are detectable with the individual components. Both the processing “intermediates” and the mature nClpP are found in assembled form.

Promoters of the Barley Germin-Like GER4 Gene Cluster Enable Strong Transgene Expression in Response to Pathogen Attack

The GER4 transcript encoding a germin-like protein of barley is derived from a dense cluster of tandemly duplicated, pathogen-induced genes that are subject to purifying selection. The GER4c promoter was found to regulate strong and pathogen-specific expression of the GUS reporter in barley, and multiple redundant WRKY factor binding sites were required for promoter activation. Immunity of plants triggered by pathogen-associated molecular patterns (PAMPs) is based on the execution of an evolutionarily conserved defense response that includes the accumulation of pathogenesis-related (PR) proteins as well as multiple other defenses. The most abundant PR transcript of barley (Hordeum vulgare) leaf epidermis attacked by the powdery mildew fungus Blumeria graminis f. sp hordei encodes the germin-like protein GER4, which has superoxide dismutase activity and functions in PAMP-triggered immunity. Here, we show that barley GER4 is encoded by a dense cluster of tandemly duplicated genes (GER4a-h) that underwent several cycles of duplication. The genomic organization of the GER4 locus also provides evidence for repeated gene birth and death cycles. The GER4 promoters contain multiple WRKY factor binding sites (W-boxes) preferentially located in promoter fragments that were exchanged between subfamily members by gene conversion. Mutational analysis of TATA-box proximal W-boxes used GER4c promoter-β-glucuronidase fusions to reveal their enhancing effects and functional redundancy on pathogen-induced promoter activity. The data suggest enhanced transcript dosage as an evolutionary driving force for the local expansion and functional redundancy of the GER4 locus. In addition, the GER4c promoter provides a tool to study signal transduction of PAMP-triggered immunity and to engineer strictly localized and pathogen-regulated disease resistance in transgenic cereal crops.