The NADH Dehydrogenase Nde1 Executes Cell Death after Integrating Signals from Metabolism and Proteostasis on the Mitochondrial Surface.

The proteolytic turnover of mitochondrial proteins is poorly understood. Here, we used a combination of dynamic isotope labeling and mass spectrometry to gain a global overview of mitochondrial protein turnover in yeast cells. Intriguingly, we found an exceptionally high turnover of the NADH dehydrogenase, Nde1. This homolog of the mammalian apoptosis inducing factor, AIF, forms two distinct topomers in mitochondria, one residing in the intermembrane space while the other spans the outer membrane and is exposed to the cytosol. The surface-exposed topomer triggers cell death in response to pro-apoptotic stimuli. The surface-exposed topomer is degraded by the cytosolic proteasome/Cdc48 system and the mitochondrial protease Yme1; however, it is strongly enriched in respiratory-deficient cells. Our data suggest that in addition to their role in electron transfer, mitochondrial NADH dehydrogenases such as Nde1 or AIF integrate signals from energy metabolism and cytosolic proteostasis to eliminate compromised cells from growing populations.

Degradation of FATTY ACID EXPORT PROTEIN1 by RHOMBOID-LIKE PROTEASE11 contributes to cold tolerance in Arabidopsis.

Plants need to acclimate to different stresses to optimize growth under unfavorable conditions. In Arabidopsis (Arabidopsis thaliana), the abundance of the chloroplast envelope protein FATTY ACID EXPORT PROTEIN1 (FAX1) decreases after the onset of low temperatures. However, how FAX1 degradation occurs and whether altered FAX1 abundance contributes to cold tolerance in plants remains unclear. The rapid cold-induced increase in RHOMBOID-LIKE PROTEASE11 (RBL11) transcript levels, the physical interaction of RBL11 with FAX1, the specific FAX1 degradation after RBL11 expression, and the absence of cold-induced FAX1 degradation in rbl11 loss-of-function mutants suggest that this enzyme is responsible for FAX1 degradation. Proteomic analyses showed that rbl11 mutants have higher levels of FAX1 and other proteins involved in membrane lipid homeostasis, suggesting that RBL11 is a key element in the remodeling of membrane properties during cold conditions. Consequently, in the cold, rbl11 mutants show a shift in lipid biosynthesis toward the eukaryotic pathway, which coincides with impaired cold tolerance. To test whether cold sensitivity is due to increased FAX1 levels, we analyzed FAX1 overexpressors. The rbl11 mutants and FAX1 overexpressor lines show superimposable phenotypic defects upon exposure to cold temperatures. Our re-sults show that the cold-induced degradation of FAX1 by RBL11 is critical for Arabidop-sis to survive cold and freezing periods.

Spliceosomal complex components are critical for adjusting the C:N balance during high-light acclimation.

Plant acclimation to an ever-changing environment is decisive for growth, reproduction, and survival. Light availability limits biomass production on both ends of the intensity spectrum. Therefore, the adjustment of plant metabolism is central to high-light (HL) acclimation, and the accumulation of photoprotective anthocyanins is commonly observed. However, mechanisms and factors regulating the HL acclimation response are less clear. Two Arabidopsis mutants of spliceosome components exhibiting a pronounced anthocyanin overaccumulation in HL were isolated from a forward genetic screen for new factors crucial for plant acclimation. Time-resolved physiological, transcriptome, and metabolome analysis revealed a vital function of the spliceosome components for rapidly adjusting gene expression and metabolism. Deficiency of INCREASED LEVEL OF POLYPLOIDY1 (ILP1), NTC-RELATED PROTEIN1 (NTR1), and PLEIOTROPIC REGULATORY LOCUS1 (PRL1) resulted in a marked overaccumulation of carbohydrates and strongly diminished amino acid biosynthesis in HL. While not generally limited in N-assimilation, ilp1, ntr1, and prl1 showed higher glutamate levels and reduced amino acid biosynthesis in HL. The comprehensive analysis reveals a function of the spliceosome components in the conditional regulation of the carbon:nitrogen balance and the accumulation of anthocyanins during HL acclimation. The importance of gene expression, metabolic regulation, and re-direction of carbon towards anthocyanin biosynthesis for HL acclimation are discussed.

Temporal classification of short time series data.

MOTIVATION: Within the frame of their genetic capacity, organisms are able to modify their molecular state to cope with changing environmental conditions or induced genetic disposition. As high throughput methods are becoming increasingly affordable, time series analysis techniques are applied frequently to study the complex dynamic interplay between genes, proteins, and metabolites at the physiological and molecular level. Common analysis approaches fail to simultaneously include (i) information about the replicate variance and (ii) the limited number of responses/shapes that a biological system is typically able to take. RESULTS: We present a novel approach to model and classify short time series signals, conceptually based on a classical time series analysis, where the dependency of the consecutive time points is exploited. Constrained spline regression with automated model selection separates between noise and signal under the assumption that highly frequent changes are less likely to occur, simultaneously preserving information about the detected variance. This enables a more precise representation of the measured information and improves temporal classification in order to identify biologically interpretable correlations among the data. AVAILABILITY AND IMPLEMENTATION: An open source F# implementation of the presented method and documentation of its usage is freely available in the TempClass repository, https://github.com/CSBiology/TempClass  [58].

PLANTdataHUB: a collaborative platform for continuous FAIR data sharing in plant research.

In modern reproducible, hypothesis-driven plant research, scientists are increasingly relying on research data management (RDM) services and infrastructures to streamline the processes of collecting, processing, sharing, and archiving research data. FAIR (i.e., findable, accessible, interoperable, and reusable) research data play a pivotal role in enabling the integration of interdisciplinary knowledge and facilitating the comparison and synthesis of a wide range of analytical findings. The PLANTdataHUB offers a solution that realizes RDM of scientific (meta)data as evolving collections of files in a directory - yielding FAIR digital objects called ARCs - with tools that enable scientists to plan, communicate, collaborate, publish, and reuse data on the same platform while gaining continuous quality control insights. The centralized platform is scalable from personal use to global communities and provides advanced federation capabilities for institutions that prefer to host their own satellite instances. This approach borrows many concepts from software development and adapts them to fit the challenges of the field of modern plant science undergoing digital transformation. The PLANTdataHUB supports researchers in each stage of a scientific project with adaptable continuous quality control insights, from the early planning phase to data publication. The central live instance of PLANTdataHUB is accessible at (https://git.nfdi4plants.org), and it will continue to evolve as a community-driven and dynamic resource that serves the needs of contemporary plant science.

Moderate high temperature is beneficial or detrimental depending on carbon availability in the green alga Chlamydomonas reinhardtii.

High temperatures impair plant growth and reduce agricultural yields, but the underlying mechanisms remain elusive. The unicellular green alga Chlamydomonas reinhardtii is an excellent model to study heat responses in photosynthetic cells due to its fast growth rate, many similarities in cellular processes to land plants, simple and sequenced genome, and ample genetic and genomics resources. Chlamydomonas grows in light by photosynthesis and with externally supplied acetate as an organic carbon source. Understanding how organic carbon sources affect heat responses is important for the algal industry but remains understudied. We cultivated wild-type Chlamydomonas under highly controlled conditions in photobioreactors at 25 °C (control), 35 °C (moderate high temperature), or 40 °C (acute high temperature) with or without constant acetate supply for 1 or 4 day. Treatment at 35 °C increased algal growth with constant acetate supply but reduced algal growth without sufficient acetate. The overlooked and dynamic effects of 35 °C could be explained by induced acetate uptake and metabolism. Heat treatment at 40 °C for more than 2 day was lethal to algal cultures with or without constant acetate supply. Our findings provide insights to understand algal heat responses and help improve thermotolerance in photosynthetic cells.

A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP-modulated NFAT signaling.

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcription factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astrocytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and destabilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteomics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regulate the NFAT signalosome and cAMP-SOCE crosstalk.

Competition co-immunoprecipitation reveals the interactors of the chloroplast CPN60 chaperonin machinery.

The functionality of all metabolic processes in chloroplasts depends on a balanced integration of nuclear- and chloroplast-encoded polypeptides into the plastid's proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding under ambient and stressful conditions, with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin of Escherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of very limited knowledge about the interactors. We employed the competition immunoprecipitation method for the identification of the chaperonin's interactors in Chlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL and the expected complex partners, we revealed numerous new interactors with high confidence. Proteins that qualify as putative substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical conformation and increased aggregation propensity. Immunoprecipitations targeted against a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.

Vernalization Alters Sink and Source Identities and Reverses Phloem Translocation from Taproots to Shoots in Sugar Beet.

During their first year of growth, overwintering biennial plants transport Suc through the phloem from photosynthetic source tissues to storage tissues. In their second year, they mobilize carbon from these storage tissues to fuel new growth and reproduction. However, both the mechanisms driving this shift and the link to reproductive growth remain unclear. During vegetative growth, biennial sugar beet (Beta vulgaris) maintains a steep Suc concentration gradient between the shoot (source) and the taproot (sink). To shift from vegetative to generative growth, they require a chilling phase known as vernalization. We studied sugar beet sink-source dynamics upon vernalization and showed that before flowering, the taproot underwent a reversal from a sink to a source of carbohydrates. This transition was induced by transcriptomic and functional reprogramming of sugar beet tissue, resulting in a reversal of flux direction in the phloem. In this transition, the vacuolar Suc importers and exporters TONOPLAST SUGAR TRANSPORTER2;1 and SUCROSE TRANSPORTER4 were oppositely regulated, leading to the mobilization of sugars from taproot storage vacuoles. Concomitant changes in the expression of floral regulator genes suggest that these processes are a prerequisite for bolting. Our data will help both to dissect the metabolic and developmental triggers for bolting and to identify potential targets for genome editing and breeding.

Peroxiredoxins couple metabolism and cell division in an ultradian cycle.

Redox cycles have been reported in ultradian, circadian and cell cycle-synchronized systems. Redox cycles persist in the absence of transcription and cyclin-CDK activity, indicating that cells harbor multiple coupled oscillators. Nonetheless, the causal relationships and molecular mechanisms by which redox cycles are embedded within ultradian, circadian or cell division cycles remain largely elusive. Yeast harbor an ultradian oscillator, the yeast metabolic cycle (YMC), which comprises metabolic/redox cycles, transcriptional cycles and synchronized cell division. Here, we reveal the existence of robust cycling of H2O2 and peroxiredoxin oxidation during the YMC and show that peroxiredoxin inactivation disrupts metabolic cycling and abolishes coupling with cell division. We find that thiol-disulfide oxidants and reductants predictably modulate the switching between different YMC metabolic states, which in turn predictably perturbs cell cycle entry and exit. We propose that oscillatory H2O2-dependent protein thiol oxidation is a key regulator of metabolic cycling and its coordination with cell division.

DeepSTABp: A Deep Learning Approach for the Prediction of Thermal Protein Stability.

Proteins are essential macromolecules that carry out a plethora of biological functions. The thermal stability of proteins is an important property that affects their function and determines their suitability for various applications. However, current experimental approaches, primarily thermal proteome profiling, are expensive, labor-intensive, and have limited proteome and species coverage. To close the gap between available experimental data and sequence information, a novel protein thermal stability predictor called DeepSTABp has been developed. DeepSTABp uses a transformer-based protein language model for sequence embedding and state-of-the-art feature extraction in combination with other deep learning techniques for end-to-end protein melting temperature prediction. DeepSTABp can predict the thermal stability of a wide range of proteins, making it a powerful and efficient tool for large-scale prediction. The model captures the structural and biological properties that impact protein stability, and it allows for the identification of the structural features that contribute to protein stability. DeepSTABp is available to the public via a user-friendly web interface, making it accessible to researchers in various fields.

Acclimation in plants - the Green Hub consortium.

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.

Complexome profiling on the Chlamydomonas lpa2 mutant reveals insights into PSII biogenesis and new PSII associated proteins.

While the composition and function of the major thylakoid membrane complexes are well understood, comparatively little is known about their biogenesis. The goal of this work was to shed more light on the role of auxiliary factors in the biogenesis of photosystem II (PSII). Here we have identified the homolog of LOW PSII ACCUMULATION 2 (LPA2) in Chlamydomonas. A Chlamydomonas reinhardtii lpa2 mutant grew slower in low light, was hypersensitive to high light, and exhibited aberrant structures in thylakoid membrane stacks. Chlorophyll fluorescence (Fv/Fm) was reduced by 38%. Synthesis and stability of newly made PSII core subunits D1, D2, CP43, and CP47 were not impaired. However, complexome profiling revealed that in the mutant CP43 was reduced to ~23% and D1, D2, and CP47 to ~30% of wild type levels. Levels of PSI and the cytochrome b6f complex were unchanged, while levels of the ATP synthase were increased by ~29%. PSII supercomplexes, dimers, and monomers were reduced to ~7%, ~26%, and ~60% of wild type levels, while RC47 was increased ~6-fold and LHCII by ~27%. We propose that LPA2 catalyses a step during PSII assembly without which PSII monomers and further assemblies become unstable and prone to degradation. The LHCI antenna was more disconnected from PSI in the lpa2 mutant, presumably as an adaptive response to reduce excitation of PSI. From the co-migration profiles of 1734 membrane-associated proteins, we identified three novel putative PSII associated proteins with potential roles in regulating PSII complex dynamics, assembly, and chlorophyll breakdown.

iMLP, a predictor for internal matrix targeting-like sequences in mitochondrial proteins.

Matrix targeting sequences (MTSs) direct proteins from the cytosol into mitochondria. Efficient targeting often relies on internal matrix targeting-like sequences (iMTS-Ls) which share structural features with MTSs. Predicting iMTS-Ls was tedious and required multiple tools and webservices. We present iMLP, a deep learning approach for the prediction of iMTS-Ls in protein sequences. A recurrent neural network has been trained to predict iMTS-L propensity profiles for protein sequences of interest. The iMLP predictor considerably exceeds the speed of existing approaches. Expanding on our previous work on iMTS-L prediction, we now serve an intuitive iMLP webservice available at http://iMLP.bio.uni-kl.de and a stand-alone command line tool for power user in addition.

Identification of Chloroplast Envelope Proteins with Critical Importance for Cold Acclimation.

The ability of plants to withstand cold temperatures relies on their photosynthetic activity. Thus, the chloroplast is of utmost importance for cold acclimation and acquisition of freezing tolerance. During cold acclimation, the properties of the chloroplast change markedly. To provide the most comprehensive view of the protein repertoire of the chloroplast envelope, we analyzed this membrane system in Arabidopsis (Arabidopsis thaliana) using mass spectrometry-based proteomics. Profiling chloroplast envelope membranes was achieved by a cross comparison of protein intensities across the plastid and the enriched membrane fraction under both normal and cold conditions. We used multivariable logistic regression to model the probabilities for the classification of an envelope localization. In total, we identified 38 envelope membrane intrinsic or associated proteins exhibiting altered abundance after cold acclimation. These proteins comprise several solute carriers, such as the ATP/ADP antiporter nucleotide transporter2 (NTT2; substantially increased abundance) or the maltose exporter MEX1 (substantially decreased abundance). Remarkably, analysis of the frost recovery of ntt loss-of-function and mex1 overexpressor mutants confirmed that the comparative proteome is well suited to identify key factors involved in cold acclimation and acquisition of freezing tolerance. Moreover, for proteins with known physiological function, we propose scenarios explaining their possible roles in cold acclimation. Furthermore, spatial proteomics introduces an additional layer of complexity and enables the identification of proteins differentially localized at the envelope membrane under the changing environmental regime.

Differential degradation of RNA species by autophagy-related pathways in Arabidopsis.

The plant vacuole recycles proteins and RNA delivered to it by autophagy. In this study, by isolating intact vacuoles from Arabidopsis plants, followed by subsequent RNA purification, and deep sequencing, we provide a comprehensive characterization of Arabidopsis vacuolar RNAome. In the vacuolar RNAome, we detected ribosomal RNAs, transfer RNAs, including those of chloroplast origin, and in addition small RNA types. As autophagy is a main mechanism for the transport of RNA to the vacuole, atg5-1 mutants deficient in autophagy were included in our analysis. We observed severely reduced amounts of most chloroplast-derived RNA species in these mutants. Comparisons with cellular RNA composition provided an indication of possible up-regulation of alternative RNA breakdown pathways. By contrast, vacuolar RNA processing and composition in plants lacking vacuolar ribonuclease 2, involved in cellular RNA homeostasis, only showed minor alterations, possibly because of the presence of further so far unknown vacuolar RNase species. Among the small RNA types, we detected mature miRNAs in all vacuolar preparations but at much lower frequency in atg5-1, raising the possibility of a biological role for vacuolar miRNAs.

Identification of small RNAs during cold acclimation in Arabidopsis thaliana.

BACKGROUND: Cold stress causes dynamic changes in gene expression that are partially caused by small non-coding RNAs since they regulate protein coding transcripts and act in epigenetic gene silencing pathways. Thus, a detailed analysis of transcriptional changes of small RNAs (sRNAs) belonging to all known sRNA classes such as microRNAs (miRNA) and small interfering RNA (siRNAs) in response to cold contributes to an understanding of cold-related transcriptome changes. RESULT: We subjected A. thaliana plants to cold acclimation conditions (4 °C) and analyzed the sRNA transcriptomes after 3 h, 6 h and 2 d. We found 93 cold responsive differentially expressed miRNAs and only 14 of these were previously shown to be cold responsive. We performed miRNA target prediction for all differentially expressed miRNAs and a GO analysis revealed the overrepresentation of miRNA-targeted transcripts that code for proteins acting in transcriptional regulation. We also identified a large number of differentially expressed cis- and trans-nat-siRNAs, as well as sRNAs that are derived from long non-coding RNAs. By combining the results of sRNA and mRNA profiling with miRNA target predictions and publicly available information on transcription factors, we reconstructed a cold-specific, miRNA and transcription factor dependent gene regulatory network. We verified the validity of links in the network by testing its ability to predict target gene expression under cold acclimation. CONCLUSION: In A. thaliana, miRNAs and sRNAs derived from cis- and trans-NAT gene pairs and sRNAs derived from lncRNAs play an important role in regulating gene expression in cold acclimation conditions. This study provides a fundamental database to deepen our knowledge and understanding of regulatory networks in cold acclimation.

The Chlamydomonas deg1c Mutant Accumulates Proteins Involved in High Light Acclimation.

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Sec-mediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knock-out mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.

The Role of Plastidic Trigger Factor Serving Protein Biogenesis in Green Algae and Land Plants.

Biochemical processes in chloroplasts are important for virtually all life forms. Tight regulation of protein homeostasis and the coordinated assembly of protein complexes, composed of both imported and locally synthesized subunits, are vital to plastid functionality. Protein biogenesis requires the action of cotranslationally acting molecular chaperones. One such chaperone is trigger factor (TF), which is known to cotranslationally bind most newly synthesized proteins in bacteria, thereby assisting their correct folding and maturation. However, how these processes are regulated in chloroplasts remains poorly understood. We report here functional investigation of chloroplast-localized TF (TIG1) in the green alga (Chlamydomonas reinhardtii) and the vascular land plant Arabidopsis (Arabidopsis thaliana). We show that chloroplastic TIG1 evolved as a specialized chaperone. Unlike other plastidic chaperones that are functionally interchangeable with their prokaryotic counterpart, TIG1 was not able to complement the broadly acting ortholog in Escherichia coli. Whereas general chaperone properties such as the prevention of aggregates or substrate recognition seems to be conserved between bacterial and plastidic TFs, plant TIG1s differed by associating with only a relatively small population of translating ribosomes. Furthermore, a reduction of plastidic TIG1 levels leads to deregulated protein biogenesis at the expense of increased translation, thereby disrupting the chloroplast energy household. This suggests a central role of TIG1 in protein biogenesis in the chloroplast.

TMEA: A Thermodynamically Motivated Framework for Functional Characterization of Biological Responses to System Acclimation.

The objective of gene set enrichment analysis (GSEA) in modern biological studies is to identify functional profiles in huge sets of biomolecules generated by high-throughput measurements of genes, transcripts, metabolites, and proteins. GSEA is based on a two-stage process using classical statistical analysis to score the input data and subsequent testing for overrepresentation of the enrichment score within a given functional coherent set. However, enrichment scores computed by different methods are merely statistically motivated and often elusive to direct biological interpretation. Here, we propose a novel approach, called Thermodynamically Motivated Enrichment Analysis (TMEA), to account for the energy investment in biological relevant processes. Therefore, TMEA is based on surprisal analysis, which offers a thermodynamic-free energy-based representation of the biological steady state and of the biological change. The contribution of each biomolecule underlying the changes in free energy is used in a Monte Carlo resampling procedure resulting in a functional characterization directly coupled to the thermodynamic characterization of biological responses to system perturbations. To illustrate the utility of our method on real experimental data, we benchmark our approach on plant acclimation to high light and compare the performance of TMEA with the most frequently used method for GSEA.