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Steroidal glycoalkaloids (SGAs) are toxic cholesterol-derived secondary metabolites present in several Solanaceae species. In potato, tuber SGA levels are for reasons of toxicity of concern in both table and starch cultivars. In the latter, SGAs bind to proteins and fibres in starch production side-streams and prevent their further uses as food and feed. To enable more sustainable uses of starch by-products, we have here reduced SGA biosynthesis in a starch potato cultivar using DNA-free CRISPR/Cas9. Six SGA genes were targeted, encoding enzymes acting either before cholesterol (SMO1-L, DWF1-L, DWF7-L), or after (16DOX, CYP88B1, TAMiso2). Editing efficiencies varied between 20% and 49%, and generated mutants were investigated under greenhouse and field conditions. Target mass-spectrometric analyses confirmed reduced SGA levels and alterations of sterol metabolism in mutated events. Plant height and tuber yield were reduced in several events, although this was not correlated to low SGA levels. Several knockout mutants had almost SGA-free leaves and tubers, the latter also under two SGA-inducing conditions. Similarly, both fibre and protein fractions isolated from side-streams in the starch production process from mutant tubers had very low SGA levels. By contrast, the corresponding wild-type SGA levels were almost 10-fold and, respectively, 40-fold higher than the recommended upper safe limit. The results demonstrate that glycoalkaloid-free mutants can be generated and grown with moderate yield reductions under both greenhouse and field conditions. This suggests a potential for sustainable production of high-value products, e.g., food-grade protein and fibre, from starch production side-streams of SGA knockout tubers.

Nitrogen (N) availability is a key determinant of plant growth and development. Here, we investigate how different N sources shape Arabidopsis thaliana root system architecture, metabolism and hormone dynamics. L-glutamine (L-GLN) significantly enhances root biomass compared to nitrate (KNO3) without compromising shoot growth. This effect emerges after 2 weeks and is independent of L-GLN's role as a carbon or ammonium source or of potential L-GLN-induced pH changes due to ammonium release, indicating a specific function of L-GLN as a N source and signaling molecule. A reverse genetic screen identified AMINO ACID PERMEASE 1 (AAP1)-mediated uptake and GLUTAMINE SYNTHETASE (GS)-dependent assimilation as essential for L-GLN-induced root biomass. In contrast, the N-sensing regulators NITRATE TRANSPORTER 1.1 (NRT1.1) and AMMONIUM TRANSPORTER (AMT) family members contribute to the differential root responses between KNO3 and L-GLN. Metabolic profiling revealed distinct amino acid signatures under these N sources, irrespective of genotype. Hormonal analyses showed that L-GLN modulates auxin homeostasis, with auxin supplementation restoring primary root growth and lateral root symmetry under L-GLN conditions. L-GLN also reconfigures cytokinin balance by elevating cZ while reducing tZ, collectively shaping root system architecture through hormone-dependent regulation. Together, these findings establish L-GLN as an integrator of N metabolism and hormone signaling in root development, highlighting its signaling capacity beyond nutrient supply and offering new perspectives for improving N use efficiency.

Urinary metabolites and their concentrations serve as biomarkers for identification of metabolic pathways that relate to specific diseases; therefore, fast and accurate quantification of the metabolites in urine is essential in health assessment and diagnosis. As many urinary metabolites are of polar nature, hydrophilic interaction liquid chromatography (HILIC) has been used over the last several years because it offers faster and more reproducible analyses compared to traditional techniques such as reversed-phase chromatography or capillary electrophoresis. In our study, we developed a HILIC method by using a 3 cm analytical column in connection with tandem mass spectrometry detection for quantification of 10 urinary metabolites including creatinine as the reference for normalization. As all tested metabolites contain ionizable functional groups, pH of the mobile phase was optimized to achieve baseline separation of 2 isomeric pairs (1-methyl-4-imidazoleacetic acid/1-methyl-5-imidazoleacetic acid and 1-methylhistidine/3-methylhistidine) and to obtain overall better separation efficiency resulting in a 7 min analysis. The developed method was validated in terms of sensitivity, carry-over, linearity, matrix effects, accuracy, and precision. The metabolite concentrations in healthy subjects determined by the developed method correspond well with the normal reference values found in the literature. Moreover, the method was tested on a small cohort of COVID-19 patients, where it enabled identification of differences in metabolite levels. Thus, the developed method has potential to be used routinely in a diagnostic field for high-throughput analysis of urine samples.

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase–cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a ‘folate trap’. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.

European canker, caused by the necrotrophic fungus Neonectria ditissima, is the most serious disease in apple production in Sweden. The disease is favored by a relatively cool and rainy climate. The canker damages have a significant economic impact due to reduced bearing surface and increased orchard management costs. The possibilities for chemical and biological control are very limited. Therefore, directed breeding for new resistant cultivars is urgently needed. Knowledge of inheritance of canker resistance and understanding of molecular mechanisms involved in resistant and susceptible responses to fungal attacks would facilitate breeding. In this study, we evaluated the tempo-spatial differences in plant-pathogen interactions in a set of partially resistant and susceptible cultivars by conducting metabolomic and lipidomic analyses. The major trends in metabolomics and lipidomic profiles were common among cultivars, irrespective of the degree of susceptibility. Several metabolites and lipids varied with time point and cultivar under N. ditissima infection. Putative key metabolites such as suberic acid and jasmonic acid were upregulated in all cultivars upon infection. Additionally, several lipids exhibited changes 30 to 45 days post-inoculation. Thus, the approach used seems to have resulted in a rich data set to be further analyzed in light of ongoing QTL-mapping efforts.

R2-like ligand-binding oxidase (R2lox) is a ferritin-like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross-link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry-based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.

Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.

Caveolae are bulb-shaped invaginations of the plasma membrane (PM) that undergo scission and fusion at the cell surface and are enriched in specific lipids. However, the influence of lipid composition on caveolae surface stability is not well described or understood. Accordingly, we inserted specific lipids into the cell PM via membrane fusion and studied their acute effects on caveolae dynamics. We demonstrate that sphingomyelin stabilizes caveolae to the cell surface, whereas cholesterol and glycosphingolipids drive caveolae scission from the PM. Although all three lipids accumulated specifically in caveolae, cholesterol and sphingomyelin were actively sequestered, whereas glycosphingolipids diffused freely. The ATPase EHD2 restricts lipid diffusion and counteracts lipid-induced scission. We propose that specific lipid accumulation in caveolae generates an intrinsically unstable domain prone to scission if not restrained by EHD2 at the caveolae neck. This work provides a mechanistic link between caveolae and their ability to sense the PM lipid composition.

This study investigates the role of the circadian clock in the regulation of gibberellin (GA) metabolism and growth in hybrid aspen (Populus tremula x P. tremuloides (Ptt)). We revealed a conserved function of the clock homolog PttEARLY BIRD1 (PttEBI1), but also implicated its rolein controlling tree growth. GA metabolite profiling and transcriptomic analysis in hybrid aspenlines with modulated expression of PttEBI1 or the core clock homologs PttLATE ELONGATED HYPOCOTYLs (PttLHYs) revealed significant changes in GA metabolism. These alterations werelinked to the differential expression of PttGA2ox8, a gene encoding an enzyme with both GA2-oxidase and GA20-oxidase activities. Our results indicate that modifications to circadian clockcomponents can significantly influence both GA metabolism and tree growth, providing potential strategies for improving tree biomass production.