Robert Hänsch

Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl).

Micronutrients are involved in all metabolic and cellular functions. Plants differ in their need for micronutrients, and we will focus here only on those elements that are generally accepted as essential for all higher plants: boron (B), chloride (Cl), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), and zinc (Zn). Several of these elements are redox-active that makes them essential as catalytically active cofactors in enzymes, others have enzyme-activating functions, and yet others fulfill a structural role in stabilizing proteins. In this review, we focus on the major functions of mineral micronutrients, mostly in cases where they were shown as constituents of proteins, making a selection and highlighting some functions in more detail.

New GATEWAY vectors for high throughput analyses of protein-protein interactions by bimolecular fluorescence complementation.

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation (BiFC) is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N- or C-terminal sub-fragment of S(CFP)3A or Venus, respectively, and enable the performance of multicolor BiFC (mcBiFC). We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.

Plant sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent non-enzymatic reaction step.

Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome.

Sulfite Reductase Defines a Newly Discovered Bottleneck for Assimilatory Sulfate Reduction and Is Essential for Growth and Development in Arabidopsis thaliana

This work examines role of sulfite reductase (SiR) in assimilatory reduction of inorganic sulfate to sulfide. Reduced sulfite reductase activity results in growth retardation and severe perturbations of sulfur, nitrogen, and carbon metabolism, demonstrating that, surprisingly, SiR plays a role in controlling flux in the assimilatory sulfate reduction pathway. The role of sulfite reductase (SiR) in assimilatory reduction of inorganic sulfate to sulfide has long been regarded as insignificant for control of flux in this pathway. Two independent Arabidopsis thaliana T-DNA insertion lines (sir1-1 and sir1-2), each with an insertion in the promoter region of SiR, were isolated. sir1-2 seedlings had 14% SiR transcript levels compared with the wild type and were early seedling lethal. sir1-1 seedlings had 44% SiR transcript levels and were viable but strongly retarded in growth. In mature leaves of sir1-1 plants, the levels of SiR transcript, protein, and enzymatic activity ranged between 17 and 28% compared with the wild type. The 28-fold decrease of incorporation of 35S label into Cys, glutathione, and protein in sir1-1 showed that the decreased activity of SiR generated a severe bottleneck in the assimilatory sulfate reduction pathway. Root sulfate uptake was strongly enhanced, and steady state levels of most of the sulfur-related metabolites, as well as the expression of many primary metabolism genes, were changed in leaves of sir1-1. Hexose and starch contents were decreased, while free amino acids increased. Inorganic carbon, nitrogen, and sulfur composition was also severely altered, demonstrating strong perturbations in metabolism that differed markedly from known sulfate deficiency responses. The results support that SiR is the only gene with this function in the Arabidopsis genome, that optimal activity of SiR is essential for normal growth, and that its downregulation causes severe adaptive reactions of primary and secondary metabolism.

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.

The role of abscisic acid and auxin in the response of poplar to abiotic stress

The plant hormones auxin and abscisic acid may at first sight appear to be a conflicting pair of plant regulators. Abscisic acid content increases during stress and protects plant water status. The content of free auxin in the developing xylem of poplar declines during stress, while auxin conjugates increase. This indicates that specific down-regulation of a signal transduction chain is important in plant adaptation to stress. Diminished auxin content may be a factor that adapts growth and wood development of poplar during adverse environmental conditions. To allow integration of environmental signals, abscisic acid and auxin must interact. Data are accumulating that abscisic acid-auxin cross-talk exists in plants. However, knowledge of the role of plant hormones in the response of trees to stress is scarce. Our data show that differences in the localisation of ABA synthesis exist between the annual, herbaceous plant Arabidopsis and the perennial woody species, poplar.

Improved enzyme production by bio‐pellets of Aspergillus niger: Targeted morphology engineering using titanate microparticles

The present study describes the design of bio‐pellet morphologies of the industrial working horse Aspergillus niger strains in submerged culture. The novel approach recruits the intended addition of titanate microparticles (TiSiO4, 8 µm) to the growth medium. As tested for two recombinant strains producing fructofuranosidase and glucoamylase, the enzyme titer by the titanate‐enhanced cultures in shake flasks was increased 3.7‐fold to 150 U/mL (for fructofuranosidase) and 9.5‐fold to 190 U/mL (for glucoamylase) as compared to the control. This could be successfully utilized for improved enzyme production in stirred tank reactors. Stimulated by the particles, the achieved final glucoamylase activity of 1,080 U/mL (fed‐batch) and 320 U/mL (batch) was sevenfold higher as compared to the conventional processes. The major reason for the enhanced production was the close association between the titanate particles and the fungal cells. Already below 2.5 g/L the micromaterial was found inside the pellets, including single particles embedded as 50–150 µm particle aggregates in the center resulting in core shell pellets. With increasing titanate levels the pellet size decreased from 1,700 µm (control) to 300 µm. Fluorescence based resolution of GFP expression revealed that the large pellets of the control were only active in a 200 µm surface layer. This matches with the critical penetration depth for nutrients and oxygen typically observed for fungal pellets. The biomass within the titanate derived fungal pellets, however, was completely active. This was due a reduced thickness of the biomass layer via smaller pellets as well as the core shell structure. Moreover, also the created loose inner pellet structure enabled a higher mass transfer and penetration depths for up to 500 µm. The creation of core‐shell pellets has not been achieved previously by the addition of microparticles, for example, made of talc or alumina. Due to this, the present work opens further possibilities to use microparticles for tailor‐made morphology design of filamentous fungi, especially for pellet based processes which have a long and strong industrial relevance for industrial production. Biotechnol. Bioeng. 2012; 109:462–471.

Fertile transgenic barley of different cultivars obtained by adjustment of bombardment conditions to tissue response

Abstract Fertile transgenic barley ( Hordeum vulgare L.) plants of five cultivars were obtained by an improved transformation protocol using different particle delivery devices. Varieties of barley which are hardly amenable for genetic transformation due to low tissue culture response were stably transformed by optimizing transformation parameters for each genotype separately according to their tissue culture response. Scutella of immature zygotic embryos were transformed with the plasmid pAHC25 carrying the selectable marker gene bar and the reporter gene uidA . Gene delivery was optimized using the uidA -expression as transient marker for the transformation efficiency and taking into account the maintenance of the regeneration capacity after the bombardment. After selection on media with bialaphos as selective agent, 40 independently transformed plants were regenerated and analyzed for functional expression of the bar and uidA gene. Molecular analysis by Southern hybridization confirmed the integration of both genes into the nuclear genome of the plants. Thirty-five T 0 -plants of five barley cultivars reached maturity so far. Analysis of the progeny confirmed the inheritance of the marker genes.

Cell-specific expression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase.

Pyrrolizidine alkaloids (PAs) are constitutive plant defense compounds with a sporadic taxonomic occurrence. The first committed step in PA biosynthesis is catalyzed by homospermidine synthase (HSS). Recent evidence confirmed that HSS evolved by gene duplication from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of the eukaryotic translation initiation factor 5A. To better understand the evolutionary relationship between these two enzymes, which are involved in completely different biological processes, we studied their tissue-specific expression. RNA-blot analysis, reverse transcriptase-PCR, and immunolocalization techniques demonstrated that DHS is constitutively expressed in shoots and roots of Senecio vernalis (Asteraceae), whereas HSS expression is root specific and restricted to distinct groups of endodermis and neighboring cortex cells located opposite to the phloem. All efforts to detect DHS by immunolocalization failed, but studies with promoter-β-glucuronidase fusions confirmed a general expression pattern, at least in young seedlings of tobacco (Nicotiana tabacum). The expression pattern for HSS differs completely from its ancestor DHS due to the adaptation of HSS to the specific requirements of PA biosynthesis.

Expression of the Arabidopsis Mutant abi1 Gene Alters Abscisic Acid Sensitivity, Stomatal Development, and Growth Morphology in Gray Poplars

The consequences of altered abscisic acid (ABA) sensitivity in gray poplar (Populus × canescens [Ait.] Sm.) development were examined by ectopic expression of the Arabidopsis (Arabidopsis thaliana) mutant abi1 (for abscisic acid insensitive1) gene. The expression resulted in an ABA-insensitive phenotype revealed by a strong tendency of abi1 poplars to wilt, impaired responsiveness of their stomata to ABA, and an ABA-resistant bud outgrowth. These plants therefore required cultivation under very humid conditions to prevent drought stress symptoms. Morphological alterations became evident when comparing abi1 poplars with poplars expressing Arabidopsis nonmutant ABI1 or wild-type plants. abi1 poplars showed increased stomatal size, enhanced shoot growth, and retarded leaf and root development. The increased stomatal size and its reversion to the size of wild-type plants by exogenous ABA indicate a role for ABA in regulating stomatal development. Enhanced shoot growth and retarded leaf and root development support the hypothesis that ABA acts independently from drought stress as a negative regulator of growth in shoots and as a positive regulator of growth in leaves and roots. In shoots, we observed an interaction of ABA with ethylene: abi1 poplars exhibited elevated ethylene production, and the ethylene perception inhibitor Ag+ antagonized the enhanced shoot growth. Thus, we provide evidence that ABA acts as negative regulator of shoot growth in nonstressed poplars by restricting ethylene production. Furthermore, we show that ABA has a role in regulating shoot branching by inhibiting lateral bud outgrowth.