Unser 10. Leibniz Plant Biochemistry Symposium am 7. und 8. Mai war ein großer Erfolg. Thematisch ging es in diesem Jahr um neue Methoden und Forschungsansätze der Naturstoffchemie. Die exzellenten Vorträge über Wirkstoffe…
Omanische Heilpflanze im Fokus der Phytochemie IPB-Wissenschaftler und Partner aus Dhofar haben jüngst die omanische Heilpflanze Terminalia dhofarica unter die phytochemische Lupe genommen. Die Pflanze ist reich an…
Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Wind, rain, herbivores, obstacles, neighbouring plants, etc. provide important mechanical cues to steerplant growth and survival. Mechanostimulation to stimulate yield and stress resistance of crops is of signifi-cant research interest, yet a molecular understanding of transcriptional responses to touch is largely absentin cereals. To address this, we performed whole-genome transcriptomics following mechanostimulation ofwheat, barley, and the recent genome-sequenced oat. The largest transcriptome changes occurred 25 minafter touching, with most of the genes being upregulated. While most genes returned to basal expressionlevel by 1–2 h in oat, many genes retained high expression even 4 h post-treatment in barley and wheat.Functional categories such as transcription factors, kinases, phytohormones, and Ca2+regulation wereaffected. In addition, cell wall-related genes involved in (hemi)cellulose, lignin, suberin, and callose biosyn-thesis were touch-responsive, providing molecular insight into mechanically induced changes in cell wallcomposition. Furthermore, several cereal-specific transcriptomic footprints were identified that were notobserved in Arabidopsis. In oat and barley, we found evidence for systemic spreading of touch-induced sig-nalling. Finally, we provide evidence that both the jasmonic acid-dependent and the jasmonic acid-independent pathways underlie touch-signalling in cereals, providing a detailed framework and markergenes for further study of (a)biotic stress responses in cereals.
Publikation
Jäckel, L.; Schnabel, A.; Stellmach, H.; Klauß, U.; Matschi, S.; Hause, G.; Vogt, T.;The terminal enzymatic step in piperine biosynthesis is co‐localized with the product piperine in specialized cells of black pepper (Piper nigrum
L.)Plant J.111731–747(2022)DOI: 10.1111/tpj.15847
Piperine (1-piperoyl piperidine) is responsible for the pungent perception of dried black pepper (Pipernigrum) fruits and essentially contributes to the aromatic properties of this spice in combination with ablend of terpenoids. The final step in piperine biosynthesis involves piperine synthase (PS), which catalyzesthe reaction of piperoyl CoA and piperidine to the biologically active and pungent amide. Nevertheless, experimental data on the cellular localization of piperine and the complete biosynthetic pathway are missing. Not only co-localization of enzymes and products, but also potential transport of piperamides to thesink organs is a possible alternative. This work, which includes purification of the native enzyme, immunolocalization, laser microdissection, fluorescence microscopy, and electron microscopy combinedwith liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), providesexperimental evidence that piperine and PS are co-localized in specialized cells of the black pepper fruit peri-sperm. PS accumulates during early stages of fruit development and its level declines before the fruits arefully mature. The product piperine is co-localized to PS and can be monitored at the cellular level by itsstrong bluish fluorescence. Rising piperine levels during fruit maturation are consistent with the increasingnumbers of fluorescent cells within the perisperm. Signal intensities of individual laser-dissected cells whenmonitored by LC-ESI-MS/MS indicate molar concentrations of this alkaloid. Significant levels of piperineand additional piperamides were also detected in cells distributed in the cortex of black pepper roots. Insummary, the data provide comprehensive experimental evidence of and insights into cell-specific biosyn-thesis and storage of piperidine alkaloids, specific and characteristic for the Piperaceae. By a combination offluorescence microscopy and LC-MS/MS analysis we localized the major piperidine alkaloids to specific cellsof the fruit perisperm and the root cortex. Immunolocalization of native piperine and piperamide synthasesshows that enzymes are co-localized with high concentrations of products in these idioblasts.
Publikation
Tannert, M.; Balcke, G. U.; Tissier, A.; Köck, M.;At4g29530 is a phosphoethanolamine phosphatase homologous to PECP1 with a role in flowering time regulationPlant J.1071072-1083(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.
Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.