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Publikation

Frey, M.; Bathe, U.; Meink, L.; Balcke, G. U.; Schmidt, J.; Frolov, A.; Soboleva, A.; Hassanin, A.; Davari, M. D.; Frank, O.; Schlagbauer, V.; Dawid, C.; Tissier, A.; Combinatorial biosynthesis in yeast leads to over 200 diterpenoids Metab. Eng. 82, 193-200, (2024) DOI: 10.1016/j.ymben.2024.02.006

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyldiphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases.
Publikation

Ai, H.; Bellstaedt, J.; Bartusch, K. S.; Eschen‐Lippold, L.; Babben, S.; Balcke, G. U.; Tissier, A.; Hause, B.; Andersen, T. G.; Delker, C.; Quint, M.; Auxin‐dependent regulation of cell division rates governs root thermomorphogenesis EMBO J. 42, e111926, (2023) DOI: 10.15252/embj.2022111926

Roots are highly plastic organs enabling plants to adapt to a changing below-ground environment. In addition to abiotic factors like nutrients or mechanical resistance, plant roots also respond to temperature variation. Below the heat stress threshold, Arabidopsis thaliana seedlings react to elevated temperature by promoting primary root growth, possibly to reach deeper soil regions with potentially better water saturation. While above-ground thermomorphogenesis is enabled by thermo-sensitive cell elongation, it was unknown how temperature modulates root growth. We here show that roots are able to sense and respond to elevated temperature independently of shoot-derived signals. This response is mediated by a yet unknown root thermosensor that employs auxin as a messenger to relay temperature signals to the cell cycle. Growth promotion is achieved primarily by increasing cell division rates in the root apical meristem, depending on de novo local auxin biosynthesis and temperature-sensitive organization of the polar auxin transport system. Hence, the primary cellular target of elevated ambient temperature differs fundamentally between root and shoot tissues, while the messenger auxin remains the same.
Publikation

Soboleva, A.; Frolova, N.; Bureiko, K.; Shumilina, J.; Balcke, G. U.; Zhukov, V. A.; Tikhonovich, I. A.; Frolov, A.; Dynamics of Reactive Carbonyl Species in Pea Root Nodules in Response to Polyethylene Glycol (PEG)-Induced Osmotic Stress Int. J. Mol. Sci. 23, 2726, (2022) DOI: 10.3390/ijms23052726

Drought dramatically affects crop productivity worldwide. For legumes this effect is especially pronounced, as their symbiotic association with rhizobia is highly-sensitive to dehydration. This might be attributed to the oxidative stress, which ultimately accompanies plants’ response to water deficit. Indeed, enhanced formation of reactive oxygen species in root nodules might result in up-regulation of lipid peroxidation and overproduction of reactive carbonyl compounds (RCCs), which readily modify biomolecules and disrupt cell functions. Thus, the knowledge of the nodule carbonyl metabolome dynamics is critically important for understanding the drought-related losses of nitrogen fixation efficiency and plant productivity. Therefore, here we provide, to the best of our knowledge, for the first time a comprehensive overview of the pea root nodule carbonyl metabolome and address its alterations in response to polyethylene glycol-induced osmotic stress as the first step to examine the changes of RCC patterns in drought treated plants. RCCs were extracted from the nodules and derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The relative quantification of CHH-derivatives by liquid chromatography-high resolution mass spectrometry with a post-run correction for derivative stability revealed in total 194 features with intensities above 1 × 105 counts, 19 of which were down- and three were upregulated. The upregulation of glyceraldehyde could accompany non-enzymatic conversion of glyceraldehyde-3-phosphate to methylglyoxal. The accumulation of 4,5-dioxovaleric acid could be the reason for down-regulation of porphyrin metabolism, suppression of leghemoglobin synthesis, inhibition of nitrogenase and degradation of legume-rhizobial symbiosis in response to polyethylene glycol (PEG)-induced osmotic stress effect. This effect needs to be confirmed with soil-based drought models.
Publikation

Tannert, M.; Balcke, G. U.; Tissier, A.; Köck, M.; At4g29530 is a phosphoethanolamine phosphatase homologous to PECP1 with a role in flowering time regulation Plant J. 107, 1072-1083, (2021) DOI: 10.1111/tpj.15367

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in membranes. The biosynthesis of phospholipids occurs mainly via the Kennedy pathway. Recent studies have shown that through this pathway, choline (Cho) moieties are synthesized through the methylation of phosphoethanolamine (PEtn) to phosphocholine (PCho) by phospho-base N-methyltransferase. In Arabidopsis thaliana, the phosphoethanolamine/phosphocholine phosphatase1 (PECP1) is described as an enzyme that regulates the synthesis of PCho by decreasing the PEtn level during phosphate starvation to avoid the energy-consuming methylation step. By homology search, we identified a gene (At4g29530) encoding a putative PECP1 homolog from Arabidopsis with a currently unknown biological function in planta. We found that At4g29530 is not induced by phosphate starvation, and is mainly expressed in leaves and flowers. The analysis of null mutants and overexpression lines revealed that PEtn, rather than PCho, is the substrate in vivo, as in PECP1. Hydrophilic interaction chromatography-coupled mass spectrometry analysis of head group metabolites shows an increased PEtn level and decreased ethanolamine level in null mutants. At4g29530 null mutants have an early flowering phenotype, which is corroborated by a higher PC/PE ratio. Furthermore, we found an increased PCho level. The choline level was not changed, so the results corroborate that the PEtn-dependent pathway is the main route for the generation of Cho moieties. We assume that the PEtn-hydrolyzing enzyme participates in fine-tuning the metabolic pathway, and helps prevent the energy-consuming biosynthesis of PCho through the methylation pathway.
Publikation

Leonova, T.; Popova, V.; Tsarev, A.; Henning, C.; Antonova, K.; Rogovskaya, N.; Vikhnina, M.; Baldensperger, T.; Soboleva, A.; Dinastia, E.; Dorn, M.; Shiroglasova, O.; Grishina, T.; Balcke, G. U.; Ihling, C.; Smolikova, G.; Medvedev, S.; Zhukov, V. A.; Babakov, V.; Tikhonovich, I. A.; Glomb, M. A.; Bilova, T.; Frolov, A.; Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds? Int. J. Mol. Sci. 21, 567, (2020) DOI: 10.3390/ijms21020567

Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.
Publikation

Sarkar, D.; Rovenich, H.; Jeena, G.; Nizam, S.; Tissier, A.; Balcke, G. U.; Mahdi, L. K.; Bonkowski, M.; Langen, G.; Zuccaro, A.; The inconspicuous gatekeeper: endophytic Serendipita vermifera acts as extended plant protection barrier in the rhizosphere New Phytol. 224, 886-901, (2019) DOI: 10.1111/nph.15904

In nature, beneficial and pathogenic fungi often simultaneously colonise plants. Despite substantial efforts to understand the composition of natural plant−microbe communities, the mechanisms driving such multipartite interactions remain largely unknown.Here we address how the interaction between the beneficial root endophyte Serendipita vermifera and the pathogen Bipolaris sorokiniana affects fungal behaviour and determines barley host responses using a gnotobiotic soil‐based split‐root system.Fungal confrontation in soil resulted in induction of B. sorokiniana genes involved in secondary metabolism and a significant repression of genes encoding putative effectors. In S. vermifera, genes encoding hydrolytic enzymes were strongly induced. This antagonistic response was not activated during the tripartite interaction in barley roots. Instead, we observed a specific induction of S. vermifera genes involved in detoxification and redox homeostasis. Pathogen infection but not endophyte colonisation resulted in substantial host transcriptional reprogramming and activation of defence. In the presence of S. vermifera, pathogen infection and disease symptoms were significantly reduced despite no marked alterations of the plant transcriptional response.The activation of stress response genes and concomitant repression of putative effector gene expression in B. sorokiniana during confrontation with the endophyte suggest a reduction of the pathogen's virulence potential before host plant infection.
Publikation

Tannert, M.; May, A.; Ditfe, D.; Berger, S.; Balcke, G. U.; Tissier, A.; Köck, M.; Pi starvation-dependent regulation of ethanolamine metabolism by phosphoethanolamine phosphatase PECP1 in Arabidopsis roots J. Exp. Bot. 69, 467-481, (2018) DOI: 10.1093/jxb/erx408

A universal plant response to phosphorus deprivation is the up-regulation of a diverse array of phosphatases. As reported recently, the AtPECP1 gene encodes a phosphatase with in vitro substrate specificity for phosphoethanolamine and phosphocholine. The putative substrates suggested that AtPECP1 is related to phospholipid metabolism; however, the biological function of AtPECP1 is as yet not understood. In addition, whereas lipid remodelling processes as part of the phosphorus starvation response have been extensively studied, knowledge of the polar head group metabolism and its regulation is lacking. We found that AtPECP1 is expressed in the cytosol and exerts by far its strongest activity in roots of phosphate-starved plants. We established a novel LC-MS/MS-based method for the quantitative and simultaneous measurement of the head group metabolites. The analysis of Atpecp1 null mutants and overexpression lines revealed that phosphoethanolamine, but not phosphocholine is the substrate of AtPECP1 in vivo. The impact on head group metabolite levels is greatest in roots of both loss-of-function and gain-of-function transgenic lines, indicating that the biological role of AtPECP1 is mainly restricted to roots. We suggest that phosphoethanolamine hydrolysis by AtPECP1 during Pi starvation is required to down-regulate the energy-consuming biosynthesis of phosphocholine through the methylation pathway.
Publikation

Stauder, R.; Welsch, R.; Camagna, M.; Kohlen, W.; Balcke, G. U.; Tissier, A.; Walter, M. H.; Strigolactone Levels in Dicot Roots Are Determined by an Ancestral Symbiosis-Regulated Clade of the PHYTOENE SYNTHASE Gene Family Front. Plant Sci. 9, 255, (2018) DOI: 10.3389/fpls.2018.00255

Strigolactones (SLs) are apocarotenoid phytohormones synthesized from carotenoid precursors. They are produced most abundantly in roots for exudation into the rhizosphere to cope with mineral nutrient starvation through support of root symbionts. Abscisic acid (ABA) is another apocarotenoid phytohormone synthesized in roots, which is involved in responses to abiotic stress. Typically low carotenoid levels in roots raise the issue of precursor supply for the biosynthesis of these two apocarotenoids in this organ. Increased ABA levels upon abiotic stress in Poaceae roots are known to be supported by a particular isoform of phytoene synthase (PSY), catalyzing the rate-limiting step in carotenogenesis. Here we report on novel PSY3 isogenes from Medicago truncatula (MtPSY3) and Solanum lycopersicum (SlPSY3) strongly expressed exclusively upon root interaction with symbiotic arbuscular mycorrhizal (AM) fungi and moderately in response to phosphate starvation. They belong to a widespread clade of conserved PSYs restricted to dicots (dPSY3) distinct from the Poaceae-PSY3s involved in ABA formation. An ancient origin of dPSY3s and a potential co-evolution with the AM symbiosis is discussed in the context of PSY evolution. Knockdown of MtPSY3 in hairy roots of M. truncatula strongly reduced SL and AM-induced C13 α-ionol/C14 mycorradicin apocarotenoids. Inhibition of the reaction subsequent to phytoene synthesis revealed strongly elevated levels of phytoene indicating induced flux through the carotenoid pathway in roots upon mycorrhization. dPSY3 isogenes are coregulated with upstream isogenes and downstream carotenoid cleavage steps toward SLs (D27, CCD7, CCD8) suggesting a combined carotenoid/apocarotenoid pathway, which provides “just in time”-delivery of precursors for apocarotenoid formation.
Publikation

Wang, J.-Z.; Li, B.; Xiao, Y.; Ni, Y.; Ke, H.; Yang, P.; de Souza, A.; Bjornson, M.; He, X.; Shen, Z.; Balcke, G. U.; Briggs, S. P.; Tissier, A.; Kliebenstein, D. J.; Dehesh, K.; Initiation of ER Body Formation and Indole Glucosinolate Metabolism by the Plastidial Retrograde Signaling Metabolite, MEcPP Mol. Plant 10, 1400-1416, (2017) DOI: 10.1016/j.molp.2017.09.012

Plants have evolved tightly regulated signaling networks to respond and adapt to environmental perturbations, but the nature of the signaling hub(s) involved have remained an enigma. We have previously established that methylerythritol cyclodiphosphate (MEcPP), a precursor of plastidial isoprenoids and a stress-specific retrograde signaling metabolite, enables cellular readjustments for high-order adaptive functions. Here, we specifically show that MEcPP promotes two Brassicaceae-specific traits, namely endoplasmic reticulum (ER) body formation and induction of indole glucosinolate (IGs) metabolism selectively, via transcriptional regulation of key regulators NAI1 for ER body formation and MYB51/122 for IGs biosynthesis). The specificity of MEcPP is further confirmed by the lack of induction of wound-inducible ER body genes as well as IGs by other altered methylerythritol phosphate pathway enzymes. Genetic analyses revealed MEcPP-mediated COI1-dependent induction of these traits. Moreover, MEcPP signaling integrates the biosynthesis and hydrolysis of IGs through induction of nitrile-specifier protein1 and reduction of the suppressor, ESM1, and production of simple nitriles as the bioactive end product. The findings position the plastidial metabolite, MEcPP, as the initiation hub, transducing signals to adjust the activity of hard-wired gene circuitry to expand phytochemical diversity and alter the associated subcellular structure required for functionality of the secondary metabolites, thereby tailoring plant stress responses.
Publikation

Frolov, A.; Bilova, T.; Paudel, G.; Berger, R.; Balcke, G. U.; Birkemeyer, C.; Wessjohann, L. A.; Early responses of mature Arabidopsis thaliana plants to reduced water potential in the agar-based polyethylene glycol infusion drought model J. Plant Physiol. 208, 70-83, (2017) DOI: 10.1016/j.jplph.2016.09.013

Drought is one of the most important environmental stressors resulting in increasing losses of crop plant productivity all over the world. Therefore, development of new approaches to increase the stress tolerance of crop plants is strongly desired. This requires precise and adequate modeling of drought stress. As this type of stress manifests itself as a steady decrease in the substrate water potential (ψw), agar plates infused with polyethylene glycol (PEG) are the perfect experimental tool: they are easy in preparation and provide a constantly reduced ψw, which is not possible in soil models. However, currently, this model is applicable only to seedlings and cannot be used for evaluation of stress responses in mature plants, which are obviously the most appropriate objects for drought tolerance research. To overcome this limitation, here we introduce a PEG-based agar infusion model suitable for 6–8-week-old A. thaliana plants, and characterize, to the best of our knowledge for the first time, the early drought stress responses of adult plants grown on PEG-infused agar. We describe essential alterations in the primary metabolome (sugars and related compounds, amino acids and polyamines) accompanied by qualitative and quantitative changes in protein patterns: up to 87 unique stress-related proteins were annotated under drought stress conditions, whereas further 84 proteins showed a change in abundance. The obtained proteome patterns differed slightly from those reported for seedlings and soil-based models.
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