Biology subjects

Kopka, J.

Publications and source records attributed to Kopka, J..

14 recordsLinked to original sources

bioRxiv2026

Pre-chlorotic Mg2+ deficiency impairs guard-cell PSII and leaf gas exchange in Vicia faba

Magnesium deficiency impairs photosynthesis and transpiration before visible chlorosis, but whether guard cells themselves are affected at this early stage remains unresolved. We combined whole-leaf gas exchange and chlorophyll fluorescence with cell-specific fluorescence measurements of individual guard cell pairs, together with elemental analysis, metabolite profiling, adenosine triphosphate quantification, and proteome profiling of guard cell-enriched material in hydroponically grown Vicia faba. Magnesium deficiency reduced magnesium concentrations in both whole leaves and guard cell-enriched fractions. Under red light, magnesium-deficient leaves showed reduced effective quantum yield of photosystem II, CO2 assimilation, transpiration, and stomatal conductance, whereas intercellular CO2 concentration remained unchanged. Individual guard cell pairs likewise showed reduced effective quantum yield of photosystem II, demonstrating that the early deficiency response extended to guard cells. Electron transport rate and non-photochemical quenching in guard cells were not significantly altered. Only a small set of metabolites in guard cell-enriched material, mainly fatty acids and citrate, responded to magnesium deficiency, whereas ATP abundance and light-induced proton extrusion remained unchanged. In contrast, the guard cell-enriched proteome showed higher abundance of photosynthesis- and translation-related proteins despite reduced guard-cell PSII efficiency. Together, these findings identify guard cells as an early responsive cell type under magnesium deficiency and show that guard-cell photochemistry is impaired before visible chlorosis, alongside reduced whole-leaf carbon gain and water loss.

physiology↗

bioRxiv2025

In vivo RUBISCO activity in Synechocystis is regulated by RuBP availability

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the main CO2-fixing enzyme on earth and entry point of carbon into the Calvin-Benson-Bassham cycle. Fueled by photosynthesis, C-assimilation by RUBISCO must be tightly controlled. RUBISCO regulation upon transition from light to darkness is not fully understood in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Synechocystis does not have a RUBISCO activase that regulates RUBISCO activity in vascular plants by removing intrinsic sugar phosphate inhibitors from its active site. Instead, the regulatory CP12 protein of Synechocystis inactivates glyceraldehyde 3-phosphate dehydrogenase (GAPDH2) and phosphoribulokinase (PRK) during darkness. This mechanism indicates metabolic regulation of RUBISCO. We investigated C-assimilation in vivo at the transition to darkness by dynamic 13CO2 labeling experiments. We monitored RUBISCO activity by 13C-incorporation into 1-C position of 3PGA. Other than the wild type, the{Delta} cp12 mutant continued to assimilate 13CO2 into 3PGA in darkness. RUBISCO abundances and specific activities were not altered in{Delta} cp12 and upon light to dark transition. CP12 was required to shut down the CBB cycle during the night. Complementation of{Delta} cp12 by native CP12 ({Delta}cp12::cp12) and CP12 with mutated conserved cysteines in its GAPDH2- and PRK-binding domains ({Delta}cp12::cp12{Delta}Cys) showed that both native binding domains are required to fully inactivate the CBB cycle in the night. RuBP levels were highly elevated in{Delta} cp12 upon transition to darkness. Complementation with mutated and native CP12 variants gradually reduced RuBP to wild type levels and revealed highly significant correlation between RuBP concentration and the time-shifted 13C-uptake into 3PGA. We propose that RUBISCO activity in Synechocystis at day-night transition is regulated through depletion and blocked regeneration of RuBP. 13C-positional analyses of aspartate suggest regeneration of RuBP in{Delta} cp12 via dysregulated gluconeogenesis and the oxidative pentose phosphate path. We demonstrate that RUBISCO activity of Synechocystis is present throughout diurnal growth and depends on the availability of its substrate.

plant biology↗

bioRxiv2025

Insights into CPSFL1 Induced Membrane Dynamics: A Multifaceted Regulator Linking Vesicle Formation to Thylakoid Biogenesis

Light drives plant life through photosynthesis, a process that takes place in the thylakoid membrane of the chloroplast, an organelle of cyanobacterial origin. The formation of thylakoid membranes within the chloroplast involves the eukaryote-specific factor CHLOROPLAST SEC14 LIKE PROTEIN 1 (CPSFL1), which shares strong sequence homology with the vesicle trafficking regulator SEC14. CSPFL1 is essential for vesicle formation, yet its specific molecular function in this process has remained unclear. In this study, we characterized CSPFL1 functions both in vitro and in vivo. Using a minimal membrane system of giant unilamellar vesicles (GUVs), we show that CPSFL1 alone can induce vesiculation. This process is mediated by lipid binding and membrane deformation, driven by curvature sensing and lipid-protein electrostatics. When expressed in the prokaryote E. coli, the eukaryote-specific CSPFL1 induces membrane curvature and vesicle formation. Plastid CPSFL1 co-purifies with vesicular structures. Lipid compositional analysis of CPSFL1-induced vesicles from bacteria reveals the presence of quinone precursors as cargo, linking CSPFL-mediated vesicle formation to prenylquinone transport. Together, our data suggest that during plant evolution, the eukaryotic vesicle formation system was co-opted for the transport of membrane integral metabolites from the inner envelope to the thylakoid membrane.

plant biology↗

bioRxiv2024

PRC2 facilitates the transition from heterotrophy to photoautotrophy during seedling emergence

Seed-to-seedling transition represents a key developmental and metabolic switch in plants. Catabolism of seed storage reserves fuels germination and early seedling emergence until photosynthesis is established. The developmental transition is controlled by Polycomb repressive complex 2 (PRC2). However, the coordination of PRC2 activity and its contribution to transcriptional reprogramming during seedling establishment is unknown. By analysing the re-distribution of H3K27me3 and changes in gene transcription in shoot and root tissues of heterotrophic and photoautotrophic seedlings, we reveal two phases of PRC2-mediated gene repression. The first phase is independent of light and photosynthesis and results in irreversible repression of the embryo maturation programme, marked by heterotrophy and biosynthesis of reserve storage molecules. The second phase is associated with the repression of metabolic pathways related to germination and early seedling emergence, and H3K27me3 deposition in this phase is sensitive to photosynthesis inhibition. We show that preventing transcription of the PRC2-repressed glyoxylate cycle gene ISOCITRATE LYASE is sufficient to drive the vegetative phase transition in PRC2-depleted plants. This underscores a key role of PRC2 repression in the coordinated metabolic and developmental switches during seedling emergence and emphasizes the close connection between metabolic and developmental identities.

plant biology↗

bioRxiv2024

Unveiling the potato cultivars with microbiome interactive traits for sustainable agricultural production

Root traits significantly shape rhizosphere microbiomes, yet their interaction with microbes is often overlooked in plant breeding programs. Here, we propose that selecting modern cultivars based on microbiome interactive traits (MITs), such as root biomass, exudate patterns and the rhizosphere microbiome, can enhance agricultural sustainability by interacting effectively with soil microbiomes, which in turn, promotes plant growth and resistance to stress, thereby reducing reliance on synthetic crop protectants. Through a stepwise selection process (in silico and in vitro) that started with approximately 1000 potato genotypes, we chose 51 potato cultivars based on known phenotypical properties and distinct root exudate patterns. We conducted a greenhouse experiment to evaluate their capacity to interact with the soil microbiome and to assess their MITs. Our findings revealed that cultivars significantly influence plant growth, metabolite profiles, and rhizosphere fungal community composition. Moreover, we observed a positive correlation between microbial community diversity and root biomass. Additionally, leaf metabolites were correlated with rhizosphere bacterial composition, supporting the plant holobiont framework. Utilising z-scores, we aggregated all data related to plant growth, metabolomes, and microbiomes, creating a classification of 51 cultivars based on a gradient of MITs. By examining the distribution of low, medium, and high MITs, we identified a group of 11 potato cultivars suitable for further studies to assess their resilience and productivity under low-input production systems. This study provides an in-depth correlation between microbiome and several plant traits across 51 cultivars, offering tools to facilitate and expedite the incorporation of microbiome traits into breeding goals to support sustainable agriculture.

microbiology↗

bioRxiv2024

Dynamic photosynthetic labelling and carbon-positional mass spectrometry monitor in vivo carbon assimilation rates by ribulose-1,5-bisphosphate carboxylase.

AbstractRibulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the most abundant enzyme and CO2 bio-sequestration system on earth. Its in vivo activity is usually determined by 14CO2 incorporation into 3-phosphoglycerate (3PGA). The radiometric analysis of 3PGA does not distinguish carbon positions. Hence, RUBISCO activity that fixes carbon into 1-C position of 3PGA and Calvin-Benson-Bassham (CBB) cycle activities that redistribute carbon into its 2-C and 3-C positions are not resolved. This study aims to provide technology that differentiates between these activities. In source fragmentation of gas chromatography-mass spectrometry (GC- MS) enables paired isotopologue distribution analyses of fragmented substructures and the complete metabolite structure. GC-MS measurements after dynamic photosynthetic 13CO2 labelling allowed quantification of the 13C fractional enrichment (E13C) and molar carbon assimilation rates (A13C) at carbon position 1-C of 3PGA by combination of E13C from carbon positions 2,3-C2 and 1,2,3-C3 with quantification of 3PGA concentrations. We validated the procedure using two GC-time of flight (TOF)-MS instruments, operated at nominal or high mass resolution and tested expected positional labelling of 3PGA by in vivo glycolysis of positional labelled glucose isotopomers. Application to{Delta} gapdh1 and{Delta} gapdh2 mutants of the highly divergent glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Synechocystis sp. PCC 6803 revealed full inactivation of the CBB cycle with maintained RUBISCO activity in the{Delta} gapdh2 mutant and a CBB cycle modulating role of GAPDH1 under fluctuating CO2 supply. RUBISCO activity in the CBB-deficient{Delta} gapdh2 mutant can re-assimilate CO2 released by catabolic pathways. We suggest that RUBISCO activity in Synechocystis can scavenge carbon loss through the pentose phosphate pathway or other cellular decarboxylation reactions.

plant biology↗

bioRxiv2024

Positional 13C Enrichment Analysis of Aspartate by GC-MS to Determine PEPC Activity In Vivo

Photoautotrophic organisms fix inorganic carbon (Ci) by two enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoenolpyruvate carboxylase (PEPC). RUBISCO assimilates Ci (CO2) into the 1-C position of 3-phosphoglycerate (3PGA). The Calvin-Benson-Basham (CBB) cycle redistributes fixed carbon atoms into 2,3-C2 of the same molecule. PEPC uses phosphoenolpyruvate (PEP) derived from 3PGA and assimilates Ci (HCO3-) into 4-C of oxaloacetate (OAA). 1,2,3-C3 of OAA and of its transaminase product aspartate originate directly from 1,2,3-C3 of 3PGA. Positional isotopologue analysis of aspartate, the main downstream metabolite of OAA in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), allows differentiation between PEPC, RUBISCO, and CBB cycle activities within one molecule. We explored in source fragmentation of gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) at nominal mass resolution and GC-atmospheric pressure chemical ionization-MS (GC-APCI-MS) at high mass resolution. This enabled the determination of fractional 13C enrichment (E13C) at each carbon position of aspartate. Two prevailing GC-MS derivatization methods, i.e. trimethylsilylation and tert-butyldimethylsilylation, were evaluated. The method was validated by 13C-isotopomer mixtures of positional labeled aspartic acid. Combination with dynamic 13CO2 labeling of Synechocystis cultures allowed direct measurements of PEPC activity in vivo alongside analyses of RUBISCO and CBB cycle activities. Accurate quantification of aspartate concentration and positional E13C provided molar Ci assimilation rates during the day and night phases of photoautotrophic Synechocystis cultures. The validated method offers several applications to characterize the photosynthetic Ci fixation in different organisms.

biochemistry↗

bioRxiv2022

Remodelled Ribosomes Synthesise a Specific Proteome in Proliferating Plant Tissue during Cold

Plant acclimation to low temperatures occurs through system-wide mechanisms that include proteome shifts, some of which occur at the level of protein synthesis. All proteins are synthesised by ribosomes. Rather than being monolithic, transcript-to-protein translation machines, ribosomes can be selective and cause effective proteome shifts required for successful temperature acclimation. Here, we use apical root meristems of germinating seedlings of the monocotyledonous plant barley as a model to study changes in protein abundance and synthesis rates during cold acclimation. We measure metabolic and physiological parameters that allow us to compare protein synthesis rates in different physiological states, e.g., in cold acclimation compared to the optimal temperature state. We show that ribosomal proteins are independently synthesised and assembled into ribosomal complexes in root proliferative tissue, and assess how the ribo-proteome shifts during cold may be associated with changes in synthesis and accumulation of macromolecular complexes. We demonstrate that translation initiation is the limiting step during cold acclimation and based on our data propose a model of a ribosomal code that depends on a reconfigured ribosome population, where as a mode of cold acclimation, specific ribosomal protein compositions may confer selective association capabilities between 60S subunits and 48S initiation complexes.

plant biology↗

bioRxiv2022

Convergence and molecular evolution of floral fragrance after independent transitions to self fertilization

The study of the independent evolution of similar characters can highlight important ecological and genetic factors that drive phenotypic evolution. The transition from reproduction by outcrossing to self-fertilization has occurred frequently throughout plant evolution. A common trend in this transition is the reduction of flower features in the selfing lineages, including display size, flower signals and pollinators rewards. These changes are believed to evolve because resources invested in building attractive flowers are reallocated to other fitness functions as the pressures to attract pollinators decrease. We investigated the similarities in the evolution of flower fragrance after independent transitions to self-fertilization in Capsella. We identified a large number of compounds that are similarly changed in different selfer lineages, such that the composition of the flower scent can predict the mating system in this genus. We further demonstrate that the emission of some of these compounds convergently evolved based on mutations in different genes. In one of the Capsella selfing lineages, the loss of {beta}-ocimene emission was caused by a mutation altering subcellular localization of the ortholog of TERPENE SYNTHASE 2 without apparent effects on its biosynthetic activity. This mutation appears to have been selected at the early stage of this selfing lineage establishment through the capture of a variant segregating in the ancestral outcrossing population. The large extent of convergence in the independent evolution of flower scent, together with the evolutionary history and molecular consequences of a causal mutation, suggest that the emission of specific volatiles has important fitness consequences in self-fertilizing plants without obvious energetic benefits.

plant biology↗

bioRxiv2021

PEPc-mediated CO2 assimilation provides carbons to gluconeogenesis and the TCA cycle in both dark-exposed and illuminated guard cells

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we used 13C-HCO3 labelling of tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling into this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results collectively suggest that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells. HighlightPEPc-mediated CO2 assimilation provides carbons for gluconeogenesis and the TCA cycle, whilst previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.

plant biology↗

bioRxiv2021

Homologs of circadian clock proteins impact the metabolic switch between light and dark growth in the cyanobacterium Synechocystis sp. PCC 6803

The putative circadian clock system of the facultative heterotrophic cyanobacterial strain Synechocystis sp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and nontargeted metabolite profiling. The strongest metabolic changes were observed in the dark in {Delta}rpaA and, to a lesser extent, in the {Delta}kaiAB1C1 mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the {Delta}rpaA mutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulator rpaA had a more pronounced effect on metabolism than the deletion of the kai genes. The larger impact of the rpaA mutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins in Synechocystis for balanced carbon and nitrogen metabolism during light-to-dark transitions.

microbiology↗

bioRxiv2021

Distinct metabolic flux modes through the tricarboxylic acid cycle in mesophyll and guard cells revealed by GC-MS-based 13C-positional isotopomer analysis

O_LI13C-Metabolic flux analysis (13C-MFA) have greatly contributed to revealing the regulation of plant metabolism. However, mass spectrometry (MS) approaches have hitherto been limited in their power to deduce flux information due to lack of positional information. C_LIO_LIHere we established an MS-based 13C-positional isotopomer labelling approach and performed a multi-species/cell-types analysis based on previous 13C-MFA to compare flux modes through the tricarboxylic acid (TCA) cycle and associated pathways in mesophyll (MCs) and guard cells (GCs). C_LIO_LIBoth cell types showed high 13C-enrichment in pyruvate. However, GCs and sink MCs, but not source MCs showed high 13C-incorporation into Glu/Gln following provision of 13C-sucrose. Only GCs showed higher 13C-enrichment in the carbon 1 atom of Gln, which is derived from PEPc-mediated CO2 fixation. Increased 13C-enrichment in the carbon 1 of Glu was also observed in both trxo1 and ntra ntrb mutants, but not in wild type Arabidopsis plants, following provision of 13C-glucose. C_LIO_LIOur results suggest that the mitochondrial thioredoxin system restricts the fluxes from PEPc and glycolysis to Glu in illuminated MCs and reveal that fluxes throughout the TCA cycle of GCs resemble those of sink MCs but operate different non-cyclic flux modes to support Gln synthesis in the light. C_LI

plant biology↗

bioRxiv2020

Arabidopsis REI-LIKE proteins activate ribosome biogenesis during cold acclimation

Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10{degrees}C, reil mutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root tissue, while tightly controlling the balance of non-translating 40S- and 60S-subunits. Reil mutations compensate by hyper-accumulation of non-translating subunits at steady-state temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that Arabidopsis buffers fluctuating translation by pre-existing non-translating ribosomes before de novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-preinitiation and pre-60S-maturation complexes and have altered paralog composition of ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, these changes are not under transcriptional control. Our study suggests requirement of de novo synthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control of NUC2 and eIF3C2 transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold acclimation. Highlight of this studyREIL proteins affect paralog composition of eukaryotic ribosomes and suppress accumulation of 43S-preinitiation and pre-60S-maturation complexes, suggesting functions of ribosome heterogeneity and biogenesis in plant cold acclimation.

plant biology↗

bioRxiv2019

The fungal endophyte Fusarium solani provokes differential effects on the energy balance of two Lotus species

Background and aimsThe interactions established between plants and endophytic fungi span a continuum from beneficial to pathogenic associations. The aim of this work was to explore the mechanisms underlying the potentially beneficial effects provoked by a fungal strain in legume species of the genus Lotus.\n\nMethodsThe ability to solubilise phosphorous was evaluated in nine fungal strains isolated from roots of L. tenuis. A selected strain was further assessed for its ability to colonize plant roots in different Lotus species. The effects of the two interactions were assessed by analysis of the photosynthesis, sugar amount, and macronutrient status of leaves and roots.\n\nResultsA fungal isolate identified as Fusarium solani shows the highest phosphate-solubilisation activity and grows endophytically in roots of L. japonicus and L. tenuis. Fungal invasion enhances plant growth in L. japonicus but provokes a contrasting effect in L. tenuis. Photosynthesis, sugars and K content showed a differential effect in both plant species.\n\nConclusionsOur results indicate neither of the plant species evaluated in this work were significantly stressed by F. solani. Thus, the differential responses observed are due to distinct mechanisms involving photosynthesis, potassium homeostasis, and carbohydrate metabolism that are employed by plants to maintain fitness during the endophytic interaction.

microbiology↗