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Publications - Cell and Metabolic Biology

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Publications

Cankar, K.; Hakkert, J. C.; Sevenier, R.; Campo, E.; Schipper, B.; Papastolopoulou, C.; Vahabi, K.; Tissier, A.; Bundock, P.; Bosch, D.; CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots Front. Plant Sci. 13, 940003, (2022) DOI: 10.3389/fpls.2022.940003

Chicory taproots accumulate sesquiterpene lactones lactucin, lactucopicrin, and 8-deoxylactucin, predominantly in their oxalated forms. The biosynthetic pathway for chicory sesquiterpene lactones has only partly been elucidated; the enzymes that convert farnesyl pyrophosphate to costunolide have been described. The next biosynthetic step of the conversion of costunolide to the tricyclic structure, guaianolide kauniolide, has so far not been elucidated in chicory. In this work three putative kauniolide synthase genes were identified in chicory named CiKLS1, CiKLS2, and CiKLS3. Their activity to convert costunolide to kauniolide was demonstrated in vitro using yeast microsome assays. Next, introduction of CRISPR/Cas9 reagents into chicory protoplasts was used to inactivate multiple chicory KLS genes and several chicory lines were successfully regenerated. The inactivation of the kauniolide synthase genes in chicory by the CRISPR/Cas9 approach resulted in interruption of the sesquiterpene lactone biosynthesis in chicory leaves and taproots. In chicory taproots, but not in leaves, accumulation of costunolide and its conjugates was observed to high levels, namely 1.5 mg/g FW. These results confirmed that all three genes contribute to STL accumulation, albeit to different extent. These observations demonstrate that three genes oriented in tandem on the chicory genome encode kauniolide synthases that initiate the conversion of costunolide toward the sesquiterpene lactones in chicory.
Publications

Abuslima, E.; Kanbar, A.; Raorane, M. L.; Eiche, E.; Junker, B. H.; Hause, B.; Riemann, M.; Nick, P.; Gain time to adapt: How sorghum acquires tolerance to salinity Front. Plant Sci. 13, 1008172, (2022) DOI: 10.3389/fpls.2022.1008172

Salinity is a global environmental threat to agricultural production and food security around the world. To delineate salt-induced damage from adaption events we analysed a pair of sorghum genotypes which are contrasting in their response to salt stress with respect to physiological, cellular, metabolomic, and transcriptional responses. We find that the salt-tolerant genotype Della can delay the transfer of sodium from the root to the shoot, more swiftly deploy accumulation of proline and antioxidants in the leaves and transfer more sucrose to the root as compared to its susceptible counterpart Razinieh. Instead Razinieh shows metabolic indicators for a higher extent photorespiration under salt stress. Following sodium accumulation by a fluorescent dye in the different regions of the root, we find that Della can sequester sodium in the vacuoles of the distal elongation zone. The timing of the adaptive responses in Della leaves indicates a rapid systemic signal from the roots that is travelling faster than sodium itself. We arrive at a model where resistance and susceptibility are mainly a matter of temporal patterns in signalling.
Publications

Grützner, R.; Schubert, R.; Horn, C.; Yang, C.; Vogt, T.; Marillonnet, S.; Engineering Betalain Biosynthesis in Tomato for High Level Betanin Production in Fruits Front. Plant Sci. 12, 682443, (2021) DOI: 10.3389/fpls.2021.682443

Betalains are pigments found in plants of the Caryophyllales order, and include the red-purple betacyanins and the yellow-orange betaxanthins. The red pigment from red beets, betanin, is made from tyrosine by a biosynthetic pathway that consists of a cytochrome P450, a L-DOPA dioxygenase, and a glucosyltransferase. The entire pathway was recently reconstituted in plants that do not make betalains naturally including potato and tomato plants. The amount of betanin produced in these plants was however not as high as in red beets. It was recently shown that a plastidic arogenate dehydrogenase gene involved in biosynthesis of tyrosine in plants is duplicated in Beta vulgaris and other betalain-producing plants, and that one of the two encoded enzymes, BvADHα, has relaxed feedback inhibition by tyrosine, contributing to the high amount of betanin found in red beets. We have reconstituted the complete betanin biosynthetic pathway in tomato plants with or without a BvADHα gene, and with all genes expressed under control of a fruit-specific promoter. The plants obtained with a construct containing BvADHα produced betanin at a higher level than plants obtained with a construct lacking this gene. These results show that use of BvADHα can be useful for high level production of betalains in heterologous hosts. Unlike red beets that produce both betacyanins and betaxanthins, the transformed tomatoes produced betacyanins only, conferring a bright purple-fuschia color to the tomato juice.
Publications

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.
Publications

Dhakarey, R.; Raorane, M. L.; Treumann, A.; Peethambaran, P. K.; Schendel, R. R.; Sahi, V. P.; Hause, B.; Bunzel, M.; Henry, A.; Kohli, A.; Riemann, M.; Corrigendum: Physiological and Proteomic Analysis of the Rice Mutant cpm2 Suggests a Negative Regulatory Role of Jasmonic Acid in Drought Tolerance Front. Plant Sci. 9, 465, (2018) DOI: 10.3389/fpls.2018.00465

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Publications

Bennewitz, S.; Bergau, N.; Tissier, A.; QTL Mapping of the Shape of Type VI Glandular Trichomes in Tomato Front. Plant Sci. 9, 1421, (2018) DOI: 10.3389/fpls.2018.01421

Glandular trichomes contribute to the high resistance of wild tomato species against insect pests not only thanks to the metabolites they produce but also because of morphological and developmental features which support the high production of these defense compounds. In Solanum habrochaites, type VI trichomes have a distinct spherical shape and a large intercellular storage cavity where metabolites can accumulate and are released upon breaking off of the glandular cells. In contrast, the type VI trichomes of S. lycopersicum have a four-leaf clover shape corresponding to the four glandular cells and a small internal cavity with limited capacity for storage of compounds. To better characterize the genetic factors underlying these trichome morphological differences we created a back-cross population of 116 individuals between S. habrochaites LA1777 and S. lycopersicum var. cerasiforme WVa106. A trichome score that reflects the shape of the type VI trichomes allowing the quantification of this trait was designed. The scores were distributed normally across the population, which was mapped with a total of 192 markers. This resulted in the identification of six quantitative trait locus (QTLs) on chromosomes I, VII, VII, and XI. The QTL on chromosome I with the highest LOD score was confirmed and narrowed down to a 500 gene interval in an advanced population derived from one of the back-cross lines. Our results provide the foundation for the genetic dissection of type VI trichome morphology and the introgression of these trichome traits into cultivated tomato lines for increased insect resistance.
Publications

Dhakarey, R.; Raorane, M. L.; Treumann, A.; Peethambaran, P. K.; Schendel, R. R.; Sahi, V. P.; Hause, B.; Bunzel, M.; Henry, A.; Kohli, A.; Riemann, M.; Physiological and Proteomic Analysis of the Rice Mutant cpm2 Suggests a Negative Regulatory Role of Jasmonic Acid in Drought Tolerance Front. Plant Sci. 8, 1903, (2017) DOI: 10.3389/fpls.2017.01903

It is widely known that numerous adaptive responses of drought-stressed plants are stimulated by chemical messengers known as phytohormones. Jasmonic acid (JA) is one such phytohormone. But there are very few reports revealing its direct implication in drought related responses or its cross-talk with other phytohormones. In this study, we compared the morpho-physiological traits and the root proteome of a wild type (WT) rice plant with its JA biosynthesis mutant coleoptile photomorphogenesis 2 (cpm2), disrupted in the allene oxide cyclase (AOC) gene, for insights into the role of JA under drought. The mutant had higher stomatal conductance, higher water use efficiency and higher shoot ABA levels under severe drought as compared to the WT. Notably, roots of cpm2 were better developed compared to the WT under both, control and drought stress conditions. Root proteome was analyzed using the Tandem Mass Tag strategy to better understand this difference at the molecular level. Expectedly, AOC was unique but notably highly abundant under drought in the WT. Identification of other differentially abundant proteins (DAPs) suggested increased energy metabolism (i.e., increased mobilization of resources) and reactive oxygen species scavenging in cpm2 under drought. Additionally, various proteins involved in secondary metabolism, cell growth and cell wall synthesis were also more abundant in cpm2 roots. Proteome-guided transcript, metabolite, and histological analyses provided further insights into the favorable adaptations and responses, most likely orchestrated by the lack of JA, in the cpm2 roots. Our results in cpm2 are discussed in the light of JA crosstalk to other phytohormones. These results together pave the path for understanding the precise role of JA during drought stress in rice.
Publications

Bergau, N.; Navarette Santos, A.; Henning, A.; Balcke, G. U.; Tissier, A.; Autofluorescence as a Signal to Sort Developing Glandular Trichomes by Flow Cytometry Front. Plant Sci. 7, 949, (2016) DOI: 10.3389/fpls.2016.00949

The industrial relevance of a number of metabolites produced in plant glandular trichomes (GTs) has spurred research on these specialized organs for a number of years. Most of the research, however, has focused on the elucidation of secondary metabolite pathways and comparatively little has been undertaken on the development and differentiation of GTs. One way to gain insight into these developmental processes is to generate stage-specific transcriptome and metabolome data. The difficulty for this resides in the isolation of early stages of development of the GTs. Here we describe a method for the separation and isolation of intact young and mature type VI trichomes from the wild tomato species Solanum habrochaites. The final and key step of the method uses cell sorting based on distinct autofluorescence signals of the young and mature trichomes. We demonstrate that sorting by flow cytometry allows recovering pure fractions of young and mature trichomes. Furthermore, we show that the sorted trichomes can be used for transcript and metabolite analyses. Because many plant tissues or cells have distinct autofluorescence components, the principles of this method can be generally applicable for the isolation of specific cell types without prior labeling.
Publications

Druege, U.; Franken, P.; Lischewski, S.; Ahkami, A. H.; Zerche, S.; Hause, B.; Hajirezaei, M. R.; Transcriptomic analysis reveals ethylene as stimulator and auxin as regulator of adventitious root formation in petunia cuttings Front. Plant Sci. 5, 494, (2014) DOI: 10.3389/fpls.2014.00494

Adventitious root (AR) formation in the stem base (SB) of cuttings is the basis for propagation of many plant species and petunia is used as model to study this developmental process. Following AR formation from 2 to 192 hours post-excision (hpe) of cuttings, transcriptome analysis by microarray revealed a change of the character of the rooting zone from SB to root identity. The greatest shift in the number of differentially expressed genes was observed between 24 and 72 hpe, when the categories storage, mineral nutrient acquisition, anti-oxidative and secondary metabolism, and biotic stimuli showed a notable high number of induced genes. Analyses of phytohormone-related genes disclosed multifaceted changes of the auxin transport system, auxin conjugation and the auxin signal perception machinery indicating a reduction in auxin sensitivity and phase-specific responses of particular auxin-regulated genes. Genes involved in ethylene biosynthesis and action showed a more uniform pattern as a high number of respective genes were generally induced during the whole process of AR formation. The important role of ethylene for stimulating AR formation was demonstrated by the application of inhibitors of ethylene biosynthesis and perception as well as of the precursor aminocyclopropane-1-carboxylic acid, all changing the number and length of AR. A model is proposed showing the putative role of polar auxin transport and resulting auxin accumulation in initiation of subsequent changes in auxin homeostasis and signal perception with a particular role of Aux/IAA expression. These changes might in turn guide the entrance into the different phases of AR formation. Ethylene biosynthesis, which is stimulated by wounding and does probably also respond to other stresses and auxin, acts as important stimulator of AR formation probably via the expression of ethylene responsive transcription factor genes, whereas the timing of different phases seems to be controlled by auxin.
Publications

Fellenberg, C.; Ziegler, J.; Handrick, V.; Vogt, T.; Polyamine homeostasis in wild type and phenolamide deficient Arabidopsis thaliana stamens Front. Plant Sci. 3, 180, (2012) DOI: 10.3389/fpls.2012.00180

Polyamines (PAs) like putrescine, spermidine, and spermine are ubiquitous polycationic molecules that occur in all living cells and have a role in a wide variety of biological processes. High amounts of spermidine conjugated to hydroxycinnamic acids are detected in the tryphine of Arabidopsis thaliana pollen grains. Tapetum localized spermidine hydroxycinnamic acid transferase (SHT) is essential for the biosynthesis of these anther specific tris-conjugated spermidine derivatives. Sht knockout lines show a strong reduction of hydroxycinnamic acid amides (HCAAs). The effect of HCAA-deficient anthers on the level of free PAs was measured by a new sensitive and reproducible method using 9-fluorenylmethyl chloroformate (FMOC) and fluorescence detection by HPLC. PA concentrations can be accurately determined even when very limited amounts of plant material, as in the case of A. thaliana stamens, are available. Analysis of free PAs in wild type stamens compared to sht deficient mutants and transcript levels of key PA biosynthetic genes revealed a highly controlled regulation of PA homeostasis in A. thaliana anthers.
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