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Publications - Cell and Metabolic Biology

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Books and chapters

Hause, B.; Yadav, H. Creation of composite plants – transformation of Medicago truncatula roots (de Bruijn, F., ed.). 1179-1184, (2020) ISBN: 9781119409144 DOI: 10.1002/9781119409144.ch152

Medicago truncatula, owing to its small diploid genome (∼500 Mbp), short life cycle, and high natural diversity makes it a good model plant and has opened the door of opportunities for scientists interested in studying legume biology. But over the years, challenges are also being faced for genetic manipulation of this plant. Many genetic manipulation protocols have been published involving Agrobacterium tumefaciens, a pathogen causing tumor disease in plants. These protocols apart from being difficult to achieve, are also time consuming. Nowadays, an easy, less time consuming and highly reproducible Agrobacterium rhizogenes based method is in use by many research groups. This method generates composite plants having transformed roots on a wild‐type shoot. Here, stable transformed lines that can be propagated over time are not achieved by this method, but for root‐development or root–microbe interaction studies this method has proven to be a useful tool for the community. In addition, transformed roots can be propagated by root organ cultures (ROCs), wherein transformed roots are propagated on sucrose containing media without any shoot part. Occasionally, even stable transgenic plants can be regenerated from transgenic roots. In this chapter, developments and improvements of various transformation protocols are discussed. The suitability of composite plants is highlighted by a study on mycorrhization of transformed and non‐transformed roots, which did not show differences in the mycorrhization rate and developmental stages of the arbuscular mycorrhizal (AM) fungus inside the roots as well as in transcript accumulation and metabolite levels of roots. Finally, applications of the A. rhizogenes based transformation method are discussed.

Ordon, J.; Bressan, M.; Kretschmer, C.; Dall’Osto, L.; Marillonnet, S.; Bassi, R.; Stuttmann, J. Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation Funct Integr Genomics 20, 151-162, (2020) DOI: 10.1007/s10142-019-00665-4

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25–30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.

El Amerany, F.; Meddich, A.; Wahbi, S.; Porzel, A.; Taourirte, M.; Rhazi, M.; Hause, B. Foliar Application of Chitosan Increases Tomato Growth and Influences Mycorrhization and Expression of Endochitinase-Encoding Genes Int J Mol Sci 21, 535, (2020) DOI: 10.3390/ijms21020535

Nowadays, applying bio-organic fertilizer (e.g., chitosan, Ch) or integrating beneficial microorganisms (e.g., arbuscular mycorrhizal fungi, AMF) are among the successful strategies to promote plant growth. Here, the effect of two application modes of Ch (foliar spray or root treatment) and Ch-derived nanoparticles (NPs) on tomato plants colonized with the AMF Rhizophagus irregularis were analyzed, thereby focusing on plant biomass, flowering and mycorrhization. An increase of shoot biomass and flower number was observed in arbuscular mycorrhizal (AM) plants sprayed with Ch. The interaction with AMF, however, was reduced as shown by decreased mycorrhization rates and AM-specific gene expression. To get insights into Ch effect on mycorrhization, levels of sugars, jasmonates, abscisic acid, and the expression of two chitinase-encoding genes were determined in mycorrhizal roots. Ch had no effect on sugar and phytohormone levels, but the reduced mycorrhization was correlated with down- and upregulated expression of Chi3 and Chi9, respectively. In contrast, application of NPs to leaves and Ch applied to the soil did not show any effect, neither on mycorrhization rate nor on growth of mycorrhizal plants. Concluding, Ch application to leaves enhanced plant growth and flowering and reduced interaction with AMF, whereas root treatment did not affect these parameters.

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.

Schulze, A.; Zimmer, M.; Mielke, S.; Stellmach, H.; Melnyk, C. W.; Hause, B.; Gasperini, D. Wound-Induced Shoot-to-Root Relocation of JA-Ile Precursors Coordinates Arabidopsis Growth Mol Plant 12, 1383-1394, (2019) DOI: 10.1016/j.molp.2019.05.013

Multicellular organisms rely on the movement of signaling molecules across cells, tissues, and organs to communicate among distal sites. In plants, localized leaf damage activates jasmonic acid (JA)-dependent transcriptional reprogramming in both harmed and unharmed tissues. Although it has been indicated that JA species can translocate from damaged into distal sites, the identity of the mobile compound(s), the tissues through which they translocate, and the effect of their relocation remain unknown. Here, we found that following shoot wounding, the relocation of endogenous jasmonates through the phloem is essential to initiate JA signaling and stunt growth in unharmed roots of Arabidopsis thaliana. By employing grafting experiments and hormone profiling, we uncovered that the hormone precursor cis-12-oxo-phytodienoic acid (OPDA) and its derivatives, but not the bioactive JA-Ile conjugate, translocate from wounded shoots into undamaged roots. Upon root relocation, the mobile precursors cooperatively regulated JA responses through their conversion into JA-Ile and JA signaling activation. Collectively, our findings demonstrate the existence of long-distance translocation of endogenous OPDA and its derivatives, which serve as mobile molecules to coordinate shoot-to-root responses, and highlight the importance of a controlled redistribution of hormone precursors among organs during plant stress acclimation.

Yadav, H.; Dreher, D.; Athmer, B.; Porzel, A.; Gavrin, A.; Baldermann, S.; Tissier, A.; Hause, B. Medicago TERPENE SYNTHASE 10 Is Involved in Defense Against an Oomycete Root Pathogen Plant Physiol 180, 1598-1613, (2019) DOI: 10.1104/pp.19.00278

In nature, plants interact with numerous beneficial or pathogenic soil-borne microorganisms. Plants have developed various defense strategies to expel pathogenic microbes, some of which function soon after pathogen infection. We used Medicago truncatula and its oomycete pathogen Aphanomyces euteiches to elucidate early responses of the infected root. A. euteiches causes root rot disease in legumes and is a limiting factor in legume production. Transcript profiling of seedlings and adult plant roots inoculated with A. euteiches zoospores for 2 h revealed specific upregulation of a gene encoding a putative sesquiterpene synthase (M. truncatula TERPENE SYNTHASE 10 [MtTPS10]) in both developmental stages. MtTPS10 was specifically expressed in roots upon oomycete infection. Heterologous expression of MtTPS10 in yeast led to production of a blend of sesquiterpenes and sesquiterpene alcohols, with NMR identifying a major peak corresponding to himalachol. Moreover, plants carrying a tobacco (Nicotiana tabacum) retrotransposon Tnt1 insertion in MtTPS10 lacked the emission of sesquiterpenes upon A. euteiches infection, supporting the assumption that the identified gene encodes a multiproduct sesquiterpene synthase. Mttps10 plants and plants with reduced MtTPS10 transcript levels created by expression of an MtTPS10-artificial microRNA in roots were more susceptible to A. euteiches infection than were the corresponding wild-type plants and roots transformed with the empty vector, respectively. Sesquiterpenes produced by expression of MtTPS10 in yeast also inhibited mycelial growth and A. euteiches zoospore germination. These data suggest that sesquiterpene production in roots by MtTPS10 plays a previously unrecognized role in the defense response of M. truncatula against A. euteiches.

Ronzan, M.; Piacentini, D.; Fattorini, L.; Federica, D. R.; Caboni, E.; Eiche, E.; Ziegler, J.; Hause, B.; Riemann, M.; Betti, C.; Altamura, M. M.; Falasca, G. Auxin-jasmonate crosstalk in Oryza sativa L. root system formation after cadmium and/or arsenic exposure Environ Exp Bot 165, 59-69, (2019) DOI: 10.1016/j.envexpbot.2019.05.013

Soil pollutants may affect root growth through interactions among phytohormones like auxin and jasmonates. Rice is frequently grown in paddy fields contaminated by cadmium and arsenic, but the effects of these pollutants on jasmonates/auxin crosstalk during adventitious and lateral roots formation are widely unknown. Therefore, seedlings of Oryza sativa cv. Nihonmasari and of the jasmonate-biosynthetic mutant coleoptile photomorphogenesis2 were exposed to cadmium and/or arsenic, and/or jasmonic acid methyl ester, and then analysed through morphological, histochemical, biochemical and molecular approaches.In both genotypes, arsenic and cadmium accumulated in roots more than shoots. In the roots, arsenic levels were more than twice higher than cadmium levels, either when arsenic was applied alone, or combined with cadmium. Pollutants reduced lateral root density in the wild -type in every treatment condition, but jasmonic acid methyl ester increased it when combined with each pollutant. Interestingly, exposure to cadmium and/or arsenic did not change lateral root density in the mutant. The transcript levels of OsASA2 and OsYUCCA2, auxin biosynthetic genes, increased in the wild-type and mutant roots when pollutants and jasmonic acid methyl ester were applied alone. Auxin (indole-3-acetic acid) levels transiently increased in the roots with cadmium and/or arsenic in the wild-type more than in the mutant. Arsenic and cadmium, when applied alone, induced fluctuations in bioactive jasmonate contents in wild-type roots, but not in the mutant. Auxin distribution was evaluated in roots of OsDR5::GUS seedlings exposed or not to jasmonic acid methyl ester added or not with cadmium and/or arsenic. The DR5::GUS signal in lateral roots was reduced by arsenic, cadmium, and jasmonic acid methyl ester. Lipid peroxidation, evaluated as malondialdehyde levels, was higher in the mutant than in the wild-type, and increased particularly in As presence, in both genotypes.Altogether, the results show that an auxin/jasmonate interaction affects rice root system development in the presence of cadmium and/or arsenic, even if exogenous jasmonic acid methyl ester only slightly mitigates pollutants toxicity.

Bergau, N.; Maul, S.; Rujescu, D.; Simm, A.; Navarrete Santos, A. Reduction of Glycolysis Intermediate Concentrations in the Cerebrospinal Fluid of Alzheimer’s Disease Patients Front Neurosci 13, 871, (2019) DOI: 10.3389/fnins.2019.00871

The profile of 122 metabolites in the cerebrospinal fluid (CSF) of patients suffering from Alzheimer’s disease (AD) and controls was studied. Among the 122 metabolites analyzed, 61 could be detected. Statistically significant differences between the AD and control group were only detected for metabolites of the glycolysis. Thus, accurate quantification of 11 glycolytic metabolites was done. We detected a significant reduction of five of them, namely phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate, pyruvate and dihydroxyacetone phosphate in the AD CSF compared to controls. These results correlate with the known reduction of glucose metabolism in the brain of patients with AD and indicate that metabolic analysis of the central carbon metabolism can be a potential tool in AD diagnostic. Although the Receiver operating characteristic (ROC) analyses of the metabolites do not reach the level of the diagnostic informativity of AD biomarkers, the combination of specific glycolysis metabolites with the established biomarkers may lead to an improvement in sensitivity and specificity.
Printed publications

Dunker, F.; Trutzenberg, A.; Rothenpieler, J. S.; Kuhn, S.; Pröls, R.; Schreiber, T.; Tissier, A.; Hückelhoven, R.; Weiberg, A. Oomycete small RNAs invade the plant RNA-induced silencing complex for virulence bioRxiv (2019) DOI: 10.1101/689190

Fungal small RNAs (sRNAs) hijack the plant RNA silencing pathway to manipulate host gene expression, named cross-kingdom RNA interference (ckRNAi). It is currently unknown how conserved and significant ckRNAi is for microbial virulence. Here, we found for the first time that sRNAs of a pathogen representing the oomycete kingdom invade the host plant’s Argonaute (AGO)/RNA-induced silencing complex. To demonstrate the functionality of the plant-invading oomycete Hyaloperonospora arabidopsidis sRNAs (HpasRNAs), we designed a novel CRISPR endoribonuclease Csy4/GUS repressor reporter to visualize in situ pathogen-induced target suppression in Arabidopsis thaliana host plant. By using 5’ RACE-PCR we demonstrated HpasRNAs-directed cleavage of plant mRNAs. The significant role of HpasRNAs together with AtAGO1 in virulence was demonstrated by plant atago1 mutants and by transgenic Arabidopsis expressing a target mimic to block HpasRNAs, that both exhibited enhanced resistance. Individual HpasRNA plant targets contributed to host immunity, as Arabidopsis gene knockout or HpasRNA-resistant gene versions exhibited quantitative enhanced or reduced susceptibility, respectively. Together with previous reports, we found that ckRNAi is conserved among oomycete and fungal pathogens.

Schubert, R.; Grunewald, S.; von Sivers, L.; Hause, B. Effects of Jasmonate on Ethylene Function during the Development of Tomato Stamens Plants 8, 277, (2019) DOI: 10.3390/plants8080277

The phenotype of the tomato mutant jasmonate-insensitive1-1 (jai1-1) mutated in the JA-Ile co-receptor COI1 demonstrates JA function in flower development, since it is female-sterile. In addition, jai1-1 exhibits a premature anther dehydration and pollen release, being in contrast to a delayed anther dehiscence in the JA-insensitive Arabidopsis mutant coi1-1. The double mutant jai1-1 Never ripe (jai1-1 Nr), which is in addition insensitive to ethylene (ET), showed a rescue of the jai1-1 phenotype regarding pollen release. This suggests that JA inhibits a premature rise in ET to prevent premature stamen desiccation. To elucidate the interplay of JA and ET in more detail, stamen development in jai1-1 Nr was compared to wild type, jai1-1 and Nr regarding water content, pollen vitality, hormone levels, and accumulation of phenylpropanoids and transcripts encoding known JA- and ET-regulated genes. For the latter, RT-qPCR based on nanofluidic arrays was employed. The data showed that additional prominent phenotypic features of jai1-1, such as diminished water content and pollen vitality, and accumulation of phenylpropanoids were at least partially rescued by the ET-insensitivity. Hormone levels and accumulation of transcripts were not affected. The data revealed that strictly JA-regulated processes cannot be rescued by ET-insensitivity, thereby emphasizing a rather minor role of ET in JA-regulated stamen development.
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