bHLH transcription factors cooperate with chromatin remodelers to regulate cell fate decisions during Arabidopsis stomatal development.

The development of multicellular organisms requires coordinated changes in gene expression that are often mediated by the interaction between transcription factors (TFs) and their corresponding cis-regulatory elements (CREs). During development and differentiation, the accessibility of CREs is dynamically modulated by the epigenome. How the epigenome, CREs, and TFs together exert control over cell fate commitment remains to be fully understood. In the Arabidopsis leaf epidermis, meristemoids undergo a series of stereotyped cell divisions, then switch fate to commit to stomatal differentiation. Newly created or reanalyzed scRNA-seq and ChIP-seq data confirm that stomatal development involves distinctive phases of transcriptional regulation and that differentially regulated genes are bound by the stomatal basic helix-loop-helix (bHLH) TFs. Targets of the bHLHs often reside in repressive chromatin before activation. MNase-seq evidence further suggests that the repressive state can be overcome and remodeled upon activation by specific stomatal bHLHs. We propose that chromatin remodeling is mediated through the recruitment of a set of physical interactors that we identified through proximity labeling-the ATPase-dependent chromatin remodeling SWI/SNF complex and the histone acetyltransferase HAC1. The bHLHs and chromatin remodelers localize to overlapping genomic regions in a hierarchical order. Furthermore, plants with stage-specific knockdown of the SWI/SNF components or HAC1 fail to activate specific bHLH targets and display stomatal development defects. Together, these data converge on a model for how stomatal TFs and epigenetic machinery cooperatively regulate transcription and chromatin remodeling during progressive fate specification.

Expanded roles and divergent regulation of FAMA in Brachypodium and Arabidopsis stomatal development.

Stomata, cellular valves found on the surfaces of aerial plant tissues, present a paradigm for studying cell fate and patterning in plants. A highly conserved core set of related basic helix-loop-helix (bHLH) transcription factors regulates stomatal development across diverse species. We characterized BdFAMA in the temperate grass Brachypodium distachyon and found this late-acting transcription factor was necessary and sufficient for specifying stomatal guard cell fate, and unexpectedly, could also induce the recruitment of subsidiary cells in the absence of its paralogue, BdMUTE. The overlap in function is paralleled by an overlap in expression pattern and by unique regulatory relationships between BdMUTE and BdFAMA. To better appreciate the relationships among the Brachypodium stomatal bHLHs, we used in vivo proteomics in developing leaves and found evidence for multiple shared interaction partners. We reexamined the roles of these genes in Arabidopsis thaliana by testing genetic sufficiency within and across species, and found that while BdFAMA and AtFAMA can rescue stomatal production in Arabidopsis fama and mute mutants, only AtFAMA can specify Brassica-specific myrosin idioblasts. Taken together, our findings refine the current models of stomatal bHLH function and regulatory feedback among paralogues within grasses as well as across the monocot/dicot divide.

Perturbations in plant energy homeostasis prime lateral root initiation via SnRK1-bZIP63-ARF19 signaling.

Plants adjust their energy metabolism to continuous environmental fluctuations, resulting in a tremendous plasticity in their architecture. The regulatory circuits involved, however, remain largely unresolved. In Arabidopsis, moderate perturbations in photosynthetic activity, administered by short-term low light exposure or unexpected darkness, lead to increased lateral root (LR) initiation. Consistent with expression of low-energy markers, these treatments alter energy homeostasis and reduce sugar availability in roots. Here, we demonstrate that the LR response requires the metabolic stress sensor kinase Snf1-RELATED-KINASE1 (SnRK1), which phosphorylates the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63) that directly binds and activates the promoter of AUXIN RESPONSE FACTOR19 (ARF19), a key regulator of LR initiation. Consistently, starvation-induced ARF19 transcription is impaired in bzip63 mutants. This study highlights a positive developmental function of SnRK1. During energy limitation, LRs are initiated and primed for outgrowth upon recovery. Hence, this study provides mechanistic insights into how energy shapes the agronomically important root system.

Advances in enzyme-mediated proximity labeling and its potential for plant research.

Cellular processes rely on the intimate interplay of different molecules, including DNA, RNA, proteins, and metabolites. Obtaining and integrating data on their abundance and dynamics at high temporal and spatial resolution are essential for our understanding of plant growth and development. In the past decade, enzymatic proximity labeling (PL) has emerged as a powerful tool to study local protein and nucleotide ensembles, discover protein-protein and protein-nucleotide interactions, and resolve questions about protein localization and membrane topology. An ever-growing number and continuous improvement of enzymes and methods keep broadening the spectrum of possible applications for PL and make it more accessible to different organisms, including plants. While initial PL experiments in plants required high expression levels and long labeling times, recently developed faster enzymes now enable PL of proteins on a cell type-specific level, even with low-abundant baits, and in different plant species. Moreover, expanding the use of PL for additional purposes, such as identification of locus-specific gene regulators or high-resolution electron microscopy may now be in reach. In this review, we give an overview of currently available PL enzymes and their applications in mammalian cell culture and plants. We discuss the challenges and limitations of PL methods and highlight open questions and possible future directions for PL in plants.

Snf1-RELATED KINASE1-Controlled C/S1-bZIP Signaling Activates Alternative Mitochondrial Metabolic Pathways to Ensure Plant Survival in Extended Darkness.

Sustaining energy homeostasis is of pivotal importance for all living organisms. In Arabidopsis thaliana, evolutionarily conserved SnRK1 kinases (Snf1-RELATED KINASE1) control metabolic adaptation during low energy stress. To unravel starvation-induced transcriptional mechanisms, we performed transcriptome studies of inducible knockdown lines and found that S1-basic leucine zipper transcription factors (S1-bZIPs) control a defined subset of genes downstream of SnRK1. For example, S1-bZIPs coordinate the expression of genes involved in branched-chain amino acid catabolism, which constitutes an alternative mitochondrial respiratory pathway that is crucial for plant survival during starvation. Molecular analyses defined S1-bZIPs as SnRK1-dependent regulators that directly control transcription via binding to G-box promoter elements. Moreover, SnRK1 triggers phosphorylation of group C-bZIPs and the formation of C/S1-heterodimers and, thus, the recruitment of SnRK1 directly to target promoters. Subsequently, the C/S1-bZIP-SnRK1 complex interacts with the histone acetylation machinery to remodel chromatin and facilitate transcription. Taken together, this work reveals molecular mechanisms underlying how energy deprivation is transduced to reprogram gene expression, leading to metabolic adaptation upon stress.

Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases.

Calcium-dependent protein kinases (CDPKs) are at the forefront of decoding transient Ca(2+) signals into physiological responses. They play a pivotal role in many aspects of plant life starting from pollen tube growth, during plant development, and in stress response to senescence and cell death. At the cellular level, Ca(2+) signals have a distinct, narrow distribution, thus requiring a conjoined localization of the decoders. Accordingly, most CDPKs have a distinct subcellular distribution which enables them to 'sense' the local Ca(2+) concentration and to interact specifically with their targets. Here we present a comprehensive overview of identified CDPK targets and discuss them in the context of kinase-substrate specificity and subcellular distribution of the CDPKs. This is particularly relevant for calcium-mediated phosphorylation where different CDPKs, as well as other kinases, were frequently reported to be involved in the regulation of the same target. However, often these studies were not performed in an in situ context. Thus, considering the specific expression patterns, distinct subcellular distribution, and different Ca(2+) affinities of CDPKs will narrow down the number of potential CDPKs for one given target. A number of aspects still remain unresolved, giving rise to pending questions for future research.

Phosphorylation of Arabidopsis transketolase at Ser428 provides a potential paradigm for the metabolic control of chloroplast carbon metabolism.

Calcium is an important second messenger in eukaryotic cells that regulates many different cellular processes. To elucidate calcium regulation in chloroplasts, we identified the targets of calcium-dependent phosphorylation within the stromal proteome. A 73 kDa protein was identified as one of the most dominant proteins undergoing phosphorylation in a calcium-dependent manner in the stromal extracts of both Arabidopsis and Pisum. It was identified as TKL (transketolase), an essential enzyme of both the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate pathway. Calcium-dependent phosphorylation of both Arabidopsis isoforms (AtTKL1 and AtTKL2) could be confirmed in vitro using recombinant proteins. The phosphorylation is catalysed by a stroma-localized protein kinase, which cannot utilize GTP. Phosphorylation of AtTKL1, the dominant isoform in most tissues, occurs at a serine residue that is conserved in TKLs of vascular plants. By contrast, an aspartate residue is present in this position in cyanobacteria, algae and mosses. Characterization of a phosphomimetic mutant (S428D) indicated that Ser428 phosphorylation exerts significant effects on the enzyme's substrate saturation kinetics at specific physiological pH values. The results of the present study point to a role for TKL phosphorylation in the regulation of carbon allocation.

Metabolism and development - integration of micro computed tomography data and metabolite profiling reveals metabolic reprogramming from floral initiation to silique development.

The interrelationship of morphogenesis and metabolism is a poorly studied phenomenon. The main paradigm is that development is controlled by gene expression. The aim of the present study was to correlate metabolism to early and late stages of flower and fruit development in order to provide the basis for the identification of metabolic adjustment and limitations. A highly detailed picture of morphogenesis is achieved using nondestructive micro computed tomography. This technique was used to quantify morphometric parameters of early and late flower development in an Arabidopsis thaliana mutant with synchronized flower initiation. The synchronized flower phenotype made it possible to sample enough early floral tissue otherwise not accessible for metabolomic analysis. The integration of metabolomic and morphometric data enabled the correlation of metabolic signatures with the process of flower morphogenesis. These signatures changed significantly during development, indicating a pronounced metabolic reprogramming in the tissue. Distinct sets of metabolites involved in these processes were identified and were linked to the findings of previous gene expression studies of flower development. High correlations with basic leucine zipper (bZIP) transcription factors and nitrogen metabolism genes involved in the control of metabolic carbon : nitrogen partitioning were revealed. Based on these observations a model for metabolic adjustment during flower development is proposed.

Spatially resolved proteomics of the Arabidopsis stomatal lineage identifies polarity complexes for cell divisions and stomatal pores.

Cell polarity is used to guide asymmetric divisions and create morphologically diverse cells. We find that two oppositely oriented cortical polarity domains present during the asymmetric divisions in the Arabidopsis stomatal lineage are reconfigured into polar domains marking ventral (pore-forming) and outward-facing domains of maturing stomatal guard cells. Proteins that define these opposing polarity domains were used as baits in miniTurboID-based proximity labeling. Among differentially enriched proteins, we find kinases, putative microtubule-interacting proteins, and polar SOSEKIs with their effector ANGUSTIFOLIA. Using AI-facilitated protein structure prediction models, we identify potential protein-protein interaction interfaces among them. Functional and localization analyses of the polarity protein OPL2 and its putative interaction partners suggest a positive interaction with mitotic microtubules and a role in cytokinesis. This combination of proteomics and structural modeling with live-cell imaging provides insights into how polarity is rewired in different cell types and cell-cycle stages.

Extensive embryonic patterning without cellular differentiation primes the plant epidermis for efficient post-embryonic stomatal activities.

Plant leaves feature epidermal stomata that are organized in stereotyped patterns. How does the pattern originate? We provide transcriptomic, imaging, and genetic evidence that Arabidopsis embryos engage known stomatal fate and patterning factors to create regularly spaced stomatal precursor cells. Analysis of embryos from 36 plant species indicates that this trait is widespread among angiosperms. Embryonic stomatal patterning in Arabidopsis is established in three stages: first, broad SPEECHLESS (SPCH) expression; second, coalescence of SPCH and its targets into discrete domains; and third, one round of asymmetric division to create stomatal precursors. Lineage progression is then halted until after germination. We show that the embryonic stomatal pattern enables fast stomatal differentiation and photosynthetic activity upon germination, but it also guides the formation of additional stomata as the leaf expands. In addition, key stomatal regulators are prevented from driving the fate transitions they can induce after germination, identifying stage-specific layers of regulation that control lineage progression during embryogenesis.

Cell Fate Programming by Transcription Factors and Epigenetic Machinery in Stomatal Development.

The development of multi-cellular organisms requires coordinated changes in gene expression that are often mediated by the interaction between transcription factors (TFs) and their corresponding cis-regulatory elements (CREs). During development and differentiation, the accessibility of CREs is dynamically modulated by the epigenome. How the epigenome, CREs and TFs together exert control over cell fate commitment remains to be fully understood. In the Arabidopsis leaf epidermis, meristemoids undergo a series of stereotyped cell divisions, then switch fate to commit to stomatal differentiation. Newly created or reanalyzed scRNA-seq and ChIP-seq data confirm that stomatal development involves distinctive phases of transcriptional regulation and that differentially regulated genes are bound by the stomatal basic-helix-loop-helix (bHLH) TFs. Targets of the bHLHs often reside in repressive chromatin before activation. MNase-seq evidence further suggests that the repressive state can be overcome and remodeled upon activation by specific stomatal bHLHs. We propose that chromatin remodeling is mediated through the recruitment of a set of physical interactors that we identified through proximity labeling - the ATPase-dependent chromatin remodeling SWI/SNF complex and the histone acetyltransferase HAC1. The bHLHs and chromatin remodelers localize to overlapping genomic regions in a hierarchical order. Furthermore, plants with stage-specific knock-down of the SWI/SNF components or HAC1 fail to activate specific bHLH targets and display stomatal development defects. Together these data converge on a model for how stomatal TFs and epigenetic machinery cooperatively regulate transcription and chromatin remodeling during progressive fate specification.

Plant organellar calcium signalling: an emerging field.

This review provides a comprehensive overview of the established and emerging roles that organelles play in calcium signalling. The function of calcium as a secondary messenger in signal transduction networks is well documented in all eukaryotic organisms, but so far existing reviews have hardly addressed the role of organelles in calcium signalling, except for the nucleus. Therefore, a brief overview on the main calcium stores in plants-the vacuole, the endoplasmic reticulum, and the apoplast-is provided and knowledge on the regulation of calcium concentrations in different cellular compartments is summarized. The main focus of the review will be the calcium handling properties of chloroplasts, mitochondria, and peroxisomes. Recently, it became clear that these organelles not only undergo calcium regulation themselves, but are able to influence the Ca(2+) signalling pathways of the cytoplasm and the entire cell. Furthermore, the relevance of recent discoveries in the animal field for the regulation of organellar calcium signals will be discussed and conclusions will be drawn regarding potential homologous mechanisms in plant cells. Finally, a short overview on bacterial calcium signalling is included to provide some ideas on the question where this typically eukaryotic signalling mechanism could have originated from during evolution.

Chloroplast-localized protein kinases: a step forward towards a complete inventory.

In addition to redox regulation, protein phosphorylation has gained increasing importance as a regulatory principle in chloroplasts in recent years. However, only very few chloroplast-localized protein kinases have been identified to date. Protein phosphorylation regulates important chloroplast processes such as photosynthesis or transcription. In order to better understand chloroplast function, it is therefore crucial to obtain a complete picture of the chloroplast kinome, which is currently constrained by two effects: first, recent observations showed that the bioinformatics-based prediction of chloroplast-localized protein kinases from available sequence data is strongly biased; and, secondly, protein kinases are of very low abundance, which makes their identification by proteomics approaches extremely difficult. Therefore, the aim of this study was to obtain a complete list of chloroplast-localized protein kinases from different species. Evaluation of protein kinases which were either highly predicted to be chloroplast localized or have been identified in different chloroplast proteomic studies resulted in the confirmation of only three new kinases. Considering also all reports of experimentally verified chloroplast protein kinases to date, compelling evidence was found for a total set of 15 chloroplast-localized protein kinases in different species. This is in contrast to a much higher number that would be expected based on targeting prediction or on the general abundance of protein kinases in relation to the entire proteome. Moreover, it is shown that unusual protein kinases with differing ATP-binding sites or catalytic centres seem to occur frequently within the chloroplast kinome, thus making their identification by mass spectrometry-based approaches even more difficult due to a different annotation.

Population pharmacokinetics and toxicodynamics of continuously infused linezolid in critically ill patients.

OBJECTIVES: Linezolid is a treatment option against multi-drug-resistant Gram-positive pathogens. Continuous infusion of linezolid has been proposed to optimize antimicrobial exposure, although pharmacokinetic data from large patient cohorts are lacking. METHODS: Population pharmacokinetics and the time-dependent association between linezolid exposure and the occurrence of thrombocytopenia in 120 critically ill patients were described. Monte Carlo simulations evaluated pharmacokinetic/pharmacodynamic/toxicodynamic target attainment in relation to body weight and creatinine clearance for continuously infused doses of 300-2400 mg/day. RESULTS: Linezolid pharmacokinetics were highly variable (interindividual variability of clearance: 52.8% coefficient of variation). Non-linear clearance was quantified, which decreased from 6.82 to 3.82 L/h within 3-6 days in the population. A relationship between linezolid exposure and platelet count over time was established. For standard dosing (1200 mg/day), the model predicted Grade 2, 3 or 4 thrombocytopenia (<75 × 103/µL, <50 × 103/µL and <25 × 103/µL) in 21.7%, 10.4% and 2.5% of patients at day 14, respectively. Patients with impaired renal function displayed higher risk. The overall probability of Grade 3 thrombocytopenia could be reduced from 10.4% using standard dosing to 6.3% if a linezolid steady state plasma concentration of 7 mg/L is targeted, suggesting a value of therapeutic drug monitoring (TDM). CONCLUSION: Dosing linezolid by continuous infusion should include considerations of creatinine clearance and body weight to maximize the achievement of therapeutic exposures. However, due to the high variability in individual dose, optimization using TDM seems necessary to optimize linezolid dosing under continuous infusion to avoid toxicity, particularly if longer treatment courses are expected.

Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID.

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

Cells contain thousands of different proteins that work together to control processes essential for life. To fully understand how these processes work it is important to know which proteins interact with each other, and which proteins are present at specific times or in certain cellular locations. Investigating this is particularly difficult if the proteins of interest are rare, either because they are present only at low levels or because they are unique to a particular type of cell. One such protein known as FAMA is only found in young guard cells in plants. Guard cells are rare cells that surround pores on the surface of leaves. They help open or close the pores to allow carbon dioxide and water in and out of the plant. Inside these cells, FAMA regulates the activity of genes in the nucleus, the compartment in the cell that houses the plant's DNA. Two recently developed molecular biology tools, called TurboID and miniTurbo, allow researchers to identify proteins that are in close contact with a protein of interest or are present at a specific place inside living animal cells. These tools use a modified enzyme to add a small chemical tag to proteins that are close to it, or anything to which it is anchored. Mair et al. adapted these tools for use in plants and tested their utility in two species that are commonly used in research: a tobacco relative called Nicotiana benthamiana, and the thale cress Arabidopsis thaliana. Their experiments showed that TurboID and miniTurbo can be used to tag proteins in different types of plant cells and organs, as well as at different stages of the plants' lives. To test whether the tools are suitable for identifying partners of rare proteins, Mair et al. used FAMA as their protein of interest. Using TurboID, they detected several proteins in close proximity to FAMA, including some that FAMA was not previously known to interact with. Mair et al. also found that TurboID could identify a number of proteins that were present in the nuclei of guard cells. This shows that the tool can be used to detect proteins in sub-compartments of rare plant cell types. Taken together, these findings show that TurboID and miniTurbo may be customized to study plant protein interactions and to explore local protein 'neighborhoods', even for rare proteins or specific cell types. To enable other plant biology researchers to easily access the TurboID and miniTurbo toolset developed in this work, it has been added to the non-profit molecular biology repository Addgene.

Redox state-dependent modulation of plant SnRK1 kinase activity differs from AMPK regulation in animals.

The evolutionarily highly conserved SNF1-related protein kinase (SnRK1) protein kinase is a metabolic master regulator in plants, balancing the critical energy consumption between growth- and stress response-related metabolic pathways. While the regulation of the mammalian [AMP-activated protein kinase (AMPK)] and yeast (SNF1) orthologues of SnRK1 is well-characterised, the regulation of SnRK1 kinase activity in plants is still an open question. Here we report that the activity and T-loop phosphorylation of AKIN10, the kinase subunit of the SnRK1 complex, is regulated by the redox status. Although this regulation is dependent on a conserved cysteine residue, the underlying mechanism is different to the redox regulation of animal AMPK and has functional implications for the regulation of the kinase complex in plants under stress conditions.

Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation.

Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.

SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants.

Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.

Solving the differential biochemical Jacobian from metabolomics covariance data.

High-throughput molecular analysis has become an integral part in organismal systems biology. In contrast, due to a missing systematic linkage of the data with functional and predictive theoretical models of the underlying metabolic network the understanding of the resulting complex data sets is lacking far behind. Here, we present a biomathematical method addressing this problem by using metabolomics data for the inverse calculation of a biochemical Jacobian matrix, thereby linking computer-based genome-scale metabolic reconstruction and in vivo metabolic dynamics. The incongruity of metabolome coverage by typical metabolite profiling approaches and genome-scale metabolic reconstruction was solved by the design of superpathways to define a metabolic interaction matrix. A differential biochemical Jacobian was calculated using an approach which links this metabolic interaction matrix and the covariance of metabolomics data satisfying a Lyapunov equation. The predictions of the differential Jacobian from real metabolomic data were found to be correct by testing the corresponding enzymatic activities. Moreover it is demonstrated that the predictions of the biochemical Jacobian matrix allow for the design of parameter optimization strategies for ODE-based kinetic models of the system. The presented concept combines dynamic modelling strategies with large-scale steady state profiling approaches without the explicit knowledge of individual kinetic parameters. In summary, the presented strategy allows for the identification of regulatory key processes in the biochemical network directly from metabolomics data and is a fundamental achievement for the functional interpretation of metabolomics data.