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Publikationen - Stoffwechsel- und Zellbiologie

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Publikation

Wessjohann, L.; Vogt, T.; Kufka, J.; Klein, R.; Prenyl- und Methyltransferasen in Natur und Synthese BIOspektrum 18, 22-25, (2012) DOI: 10.1007/s12268-012-0137-4

Late stage enzymatic prenylation and methylation are means to diversify (natural) compounds and to specify their functions. In eukaryotes and microbes, these steps are performed by large enzyme families, the prenyl and methyl transferases, which modify various types of small molecules, like isoprenoids, phenolics or alkaloids, but also DNA and proteins. We investigate the theoretical basis of these processes and possible commercial applications in synthetic chemistry.
Publikation

Werner, S.; Engler, C.; Weber, E.; Gruetzner, R.; Marillonnet, S.; Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system Bioengineered 3, 38-43, (2012) DOI: 10.4161/bbug.3.1.18223

Recent progress in the field of synthetic biology has led to the creation of cells containing synthetic genomes. Although these first synthetic organisms contained copies of natural genomes, future work will be directed toward engineering of organisms with modified genomes and novel phenotypes. Much work, however, remains to be done to be able to routinely engineer novel biological functions. As a tool that will be useful for such purpose, we have recently developed a modular cloning system (MoClo) that allows high throughput assembly of multiple genetic elements. We present here new features of this cloning system that allow to increase the speed of assembly of multigene constructs. As an example, 68 DNA fragments encoding basic genetic elements were assembled using three one-pot cloning steps, resulting in a 50 kb construct containing 17 eukaryotic transcription units. This cloning system should be useful for generating the multiple construct variants that will be required for developing gene networks encoding novel functions, and fine-tuning the expression levels of the various genes involved.
Publikation

Wasternack, C.; Goetz, S.; Hellwege, A.; Forner, S.; Strnad, M.; Hause, B.; Another JA/COI1-independent role of OPDA detected in tomato embryo development Plant Signal Behav. 7, 1349-1353, (2012) DOI: 10.4161/psb.21551

Jasmonates (JAs) are ubiquitously occurring signaling compounds in plants formed in response to biotic and abiotic stress as well as in development. (+)-7-iso-jasmonoyl isoleucine, the bioactive JA, is involved in most JA-dependent processes mediated by the F-box protein COI1 in a proteasome-dependent manner. However, there is an increasing number of examples, where the precursor of JA biosynthesis, cis-(+)-12-oxophytodienoic acid (OPDA) is active in a JA/COI1-independent manner. Here, we discuss those OPDA-dependent processes, thereby giving emphasis on tomato embryo development. Recent data on seed coat-generated OPDA and its role in embryo development is discussed based on biochemical and genetic evidences.
Publikation

Vadassery, J.; Reichelt, M.; Hause, B.; Gershenzon, J.; Boland, W.; Mithöfer, A.; CML42-Mediated Calcium Signaling Coordinates Responses to Spodoptera Herbivory and Abiotic Stresses in Arabidopsis Plant Physiol. 159, 1159-1175, (2012) DOI: 10.1104/pp.112.198150

In the interaction between Arabidopsis (Arabidopsis thaliana) and the generalist herbivorous insect Spodoptera littoralis, little is known about early events in defense signaling and their link to downstream phytohormone pathways. S. littoralis oral secretions induced both Ca2+ and phytohormone elevation in Arabidopsis. Plant gene expression induced by oral secretions revealed up-regulation of a gene encoding a calmodulin-like protein, CML42. Functional analysis of cml42 plants revealed more resistance to herbivory than in the wild type, because caterpillars gain less weight on the mutant, indicating that CML42 negatively regulates plant defense; cml42 also showed increased aliphatic glucosinolate content and hyperactivated transcript accumulation of the jasmonic acid (JA)-responsive genes VSP2 and Thi2.1 upon herbivory, which might contribute to increased resistance. CML42 up-regulation is negatively regulated by the jasmonate receptor Coronatine Insensitive1 (COI1), as loss of functional COI1 resulted in prolonged CML42 activation. CML42 thus acts as a negative regulator of plant defense by decreasing COI1-mediated JA sensitivity and the expression of JA-responsive genes and is independent of herbivory-induced JA biosynthesis. JA-induced Ca2+ elevation and root growth inhibition were more sensitive in cml42, also indicating higher JA perception. Our results indicate that CML42 acts as a crucial signaling component connecting Ca2+ and JA signaling. CML42 is localized to cytosol and nucleus. CML42 is also involved in abiotic stress responses, as kaempferol glycosides were down-regulated in cml42, and impaired in ultraviolet B resistance. Under drought stress, the level of abscisic acid accumulation was higher in cml42 plants. Thus, CML42 might serve as a Ca2+ sensor having multiple functions in insect herbivory defense and abiotic stress responses.
Publikation

Tissier, A.; Glandular trichomes: what comes after expressed sequence tags? Plant J. 70, 51-68, (2012) DOI: 10.1111/j.1365-313X.2012.04913.x

Glandular trichomes cover the surface of many plant species. They exhibit tremendous diversity, be it in their shape or the compounds they secrete. This diversity is expressed between species but also within species or even individual plants. The industrial uses of some trichome secretions and their potential as a defense barrier, for example against arthropod pests, has spurred research into the biosynthesis pathways that lead to these specialized metabolites. Because complete biosynthesis pathways take place in the secretory cells, the establishment of trichome‐specific expressed sequence tag libraries has greatly accelerated their elucidation. Glandular trichomes also have an important metabolic capacity and may be considered as true cell factories. To fully exploit the potential of glandular trichomes as breeding or engineering objects, several research areas will have to be further investigated, such as development, patterning, metabolic fluxes and transcription regulation. The purpose of this review is to provide an update on the methods and technologies which have been used to investigate glandular trichomes and to propose new avenues of research to deepen our understanding of these specialized structures.
Publikation

Stenzel, I.; Otto, M.; Delker, C.; Kirmse, N.; Schmidt, D.; Miersch, O.; Hause, B.; Wasternack, C.; ALLENE OXIDE CYCLASE (AOC) gene family members of Arabidopsis thaliana: tissue- and organ-specific promoter activities and in vivo heteromerization J. Exp. Bot. 63, 6125-6138, (2012) DOI: 10.1093/jxb/ers261

Jasmonates are important signals in plant stress responses and plant development. An essential step in the biosynthesis of jasmonic acid (JA) is catalysed by ALLENE OXIDE CYCLASE (AOC) which establishes the naturally occurring enantiomeric structure of jasmonates. In Arabidopsis thaliana, four genes encode four functional AOC polypeptides (AOC1, AOC2, AOC3, and AOC4) raising the question of functional redundancy or diversification. Analysis of transcript accumulation revealed an organ-specific expression pattern, whereas detailed inspection of transgenic lines expressing the GUS reporter gene under the control of individual AOC promoters showed partially redundant promoter activities during development: (i) In fully developed leaves, promoter activities of AOC1, AOC2, and AOC3 appeared throughout all leaf tissue, but AOC4 promoter activity was vascular bundle-specific; (ii) only AOC3 and AOC4 showed promoter activities in roots; and (iii) partially specific promoter activities were found for AOC1 and AOC4 in flower development. In situ hybridization of flower stalks confirmed the GUS activity data. Characterization of single and double AOC loss-of-function mutants further corroborates the hypothesis of functional redundancies among individual AOCs due to a lack of phenotypes indicative of JA deficiency (e.g. male sterility). To elucidate whether redundant AOC expression might contribute to regulation on AOC activity level, protein interaction studies using bimolecular fluorescence complementation (BiFC) were performed and showed that all AOCs can interact among each other. The data suggest a putative regulatory mechanism of temporal and spatial fine-tuning in JA formation by differential expression and via possible heteromerization of the four AOCs.
Publikation

Sallaud, C.; Giacalone, C.; Töpfer, R.; Goepfert, S.; Bakaher, N.; Rösti, S.; Tissier, A.; Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes Plant J. 72, 1-17, (2012) DOI: 10.1111/j.1365-313X.2012.05068.x

Leaves of tobacco (Nicotiana tabacum) are covered with glandular trichomes that produce sucrose esters and diterpenoids in varying quantities, depending on cultivar type. The bicyclic diterpene Z‐abienol is the major labdanoid present in some oriental tobacco cultivars, where it constitutes a precursor of important flavours and aromas. We describe here the identification and characterization of two genes governing the biosynthesis of Z‐abienol in N. tabacum. As for other angiosperm labdanoid diterpenes, the biosynthesis of Z‐abienol proceeds in two steps. NtCPS2 encodes a class‐II terpene synthase that synthesizes 8‐hydroxy‐copalyl diphosphate, and NtABS encodes a kaurene synthase‐like (KSL) protein that uses 8‐hydroxy‐copalyl diphosphate to produce Z‐abienol. Phylogenetic analysis indicates that NtABS belongs to a distinct clade of KSL proteins that comprises the recently identified tomato (Solanum habrochaites) santalene and bergamotene synthase. RT‐PCR results show that both genes are preferentially expressed in trichomes. Moreover, microscopy of NtCPS2 promoter‐GUS fusion transgenics demonstrated a high specificity of expression to trichome glandular cells. Ectopic expression of both genes, but not of either one alone, driven by a trichome‐specific promoter in transgenic Nicotiana sylvestris conferred Z‐abienol formation to this species, which does not normally produce it. Furthermore, sequence analysis of over 100 tobacco cultivars revealed polymorphisms in NtCPS2 that lead to a prematurely truncated protein in cultivars lacking Z‐abienol, thus establishing NtCPS2 as a major gene controlling Z‐abienol biosynthesis in tobacco. These results offer new perspectives for tobacco breeding and the metabolic engineering of labdanoid diterpenes, as well as for structure–function relationship studies of terpene synthases.
Publikation

Landgraf, R.; Schaarschmidt, S.; Hause, B.; Repeated leaf wounding alters the colonization of Medicago truncatula roots by beneficial and pathogenic microorganisms Plant Cell Environ. 35, 1344-1357, (2012) DOI: 10.1111/j.1365-3040.2012.02495.x

In nature, plants are subject to various stresses that are often accompanied by wounding of the aboveground tissues. As wounding affects plants locally and systemically, we investigated the impact of leaf wounding on interactions of Medicago truncatula with root‐colonizing microorganisms, such as the arbuscular mycorrhizal (AM) fungus Glomus intraradices, the pathogenic oomycete Aphanomyces euteiches and the nitrogen‐fixing bacterium Sinorhizobium meliloti. To obtain a long‐lasting wound response, repeated wounding was performed and resulted in locally and systemically increased jasmonic acid (JA) levels accompanied by the expression of jasmonate‐induced genes, among them the genes encoding allene oxide cyclase 1 (MtAOC1) and a putative cell wall‐bound invertase (cwINV). After repeated wounding, colonization with the AM fungus was increased, suggesting a role of jasmonates as positive regulators of mycorrhization, whereas the interaction with the rhizobacterium was not affected. In contrast, wounded plants appeared to be less susceptible to pathogens which might be caused by JA‐induced defence mechanisms. The effects of wounding on mycorrhization and pathogen infection could be partially mimicked by foliar application of JA. In addition to JA itself, the positive effect on mycorrhization might be mediated by systemically induced cwINV, which was previously shown to exhibit a regulatory function on interaction with AM fungi.
Publikation

Goetz, S.; Hellwege, A.; Stenzel, I.; Kutter, C.; Hauptmann, V.; Forner, S.; McCaig, B.; Hause, G.; Miersch, O.; Wasternack, C.; Hause, B.; Role of cis-12-Oxo-Phytodienoic Acid in Tomato Embryo Development Plant Physiol. 158, 1715-1727, (2012) DOI: 10.1104/pp.111.192658

Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.
Publikation

Gaupels, F.; Sarioglu, H.; Beckmann, M.; Hause, B.; Spannagl, M.; Draper, J.; Lindermayr, C.; Durner, J.; Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling Plant Physiol. 160, 2285-2299, (2012) DOI: 10.1104/pp.112.205336

In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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