Functional in vitro diversity of an intrinsically disordered plant protein during freeze-thawing is encoded by its structural plasticity.

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

Two mitochondrial phosphatases, PP2c63 and Sal2, are required for posttranslational regulation of the TCA cycle in Arabidopsis.

Protein phosphorylation is a well-established post-translational mechanism that regulates protein functions and metabolic pathways. It is known that several plant mitochondrial proteins are phosphorylated in a reversible manner. However, the identities of the protein kinases/phosphatases involved in this mechanism and their roles in the regulation of the tricarboxylic acid (TCA) cycle remain unclear. In this study, we isolated and characterized plants lacking two mitochondrially targeted phosphatases (Sal2 and PP2c63) along with pyruvate dehydrogenase kinase (PDK). Protein-protein interaction analysis, quantitative phosphoproteomics, and enzymatic analyses revealed that PDK specifically regulates pyruvate dehydrogenase complex (PDC), while PP2c63 nonspecifically regulates PDC. When recombinant PP2c63 and Sal2 proteins were added to mitochondria isolated from mutant plants, protein-protein interaction and enzymatic analyses showed that PP2c63 directly phosphorylates and modulates the activity of PDC, while Sal2 only indirectly affects TCA cycle enzymes. Characterization of steady-state metabolite levels and fluxes in the mutant lines further revealed that these phosphatases regulate flux through the TCA cycle, and that altered metabolism in the sal2 pp2c63 double mutant compromises plant growth. These results are discussed in the context of current models of the control of respiration in plants.

Multiplexed Profiling and Data Processing Methods to Identify Temperature-Regulated Primary Metabolites Using Gas Chromatography Coupled to Mass Spectrometry.

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of plant material that was exposed to temperature stress. The method can be combined with stable isotope labeling and tracing experiments and is equally applicable to preparations of plant material and microbial photosynthetic organisms. The described methods are modular and can be multiplexed, that is, the same sample or a paired identical backup sample can be analyzed sequentially by more than one of the described procedures. The modules include rapid sampling and metabolic inactivation protocols for samples in a wide weight range, sample extraction procedures, chemical derivatization steps that are required to make the metabolite fraction amenable to gas chromatographic analysis, routine GC-MS methods, and procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The methods have two applications: (1) The rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification by exact quantification using GC-MS protocols that are extended by internal and external standardization.

Non-aqueous Fractionation (NAF) for Metabolite Analysis in Subcellular Compartments of Arabidopsis Leaf Tissues.

The accurate determination of metabolite distribution in subcellular compartments is still challenging in plant science. Various methodologies, such as fluorescence resonance energy transfer-based technology, nuclear magnetic resonance spectroscopy and protoplast fractionation allow the study of metabolite compartmentation. However, large changes in metabolite levels occur during such procedures. Therefore, the non-aqueous fractionation (NAF) technique is currently the best method for the study of in-vivo metabolite distribution. Our protocol presents a detailed workflow including the NAF procedure and quantification of compartment-specific markers for three subcellular compartments: ADP glucose pyrophosphorylase (AGPase) as plastidic marker, phosphoenolpyruvate carboxylase (PEPC) as cytosolic marker, and nitrate and acid invertase as vacuolar markers.

Target of Rapamycin Inhibition in Chlamydomonas reinhardtii Triggers de Novo Amino Acid Synthesis by Enhancing Nitrogen Assimilation.

The Target of Rapamycin (TOR) kinase is a central regulator of growth and metabolism in all eukaryotic organisms, including animals, fungi, and plants. Even though the inputs and outputs of TOR signaling are well characterized for animals and fungi, our understanding of the upstream regulators of TOR and its downstream targets is still fragmentary in photosynthetic organisms. In this study, we employed the rapamycin-sensitive green alga Chlamydomonas reinhardtii to elucidate the molecular cause of the amino acid accumulation that occurs after rapamycin-induced inhibition of TOR. Using different growth conditions and stable 13C- and 15N-isotope labeling, we show that this phenotype is accompanied by increased nitrogen (N) uptake, which is induced within minutes of TOR inhibition. Interestingly, this increased N influx is accompanied by increased activities of glutamine synthetase and glutamine oxoglutarate aminotransferase, the main N-assimilating enzymes, which are responsible for the rise in levels of several amino acids, which occurs within a few minutes. Accordingly, we conclude that even though translation initiation and autophagy have been reported to be the main downstream targets of TOR, the upregulation of de novo amino acid synthesis seems to be one of the earliest responses induced after the inhibition of TOR in Chlamydomonas.

Rapid transcriptional and metabolic regulation of the deacclimation process in cold acclimated Arabidopsis thaliana.

BACKGROUND: During low temperature exposure, temperate plant species increase their freezing tolerance in a process termed cold acclimation. This is accompanied by dampened oscillations of circadian clock genes and disrupted oscillations of output genes and metabolites. During deacclimation in response to warm temperatures, cold acclimated plants lose freezing tolerance and resume growth and development. While considerable effort has been directed toward understanding the molecular and metabolic basis of cold acclimation, much less information is available about the regulation of deacclimation. RESULTS: We report metabolic (gas chromatography-mass spectrometry) and transcriptional (microarrays, quantitative RT-PCR) responses underlying deacclimation during the first 24 h after a shift of Arabidopsis thaliana (Columbia-0) plants cold acclimated at 4 °C back to warm temperature (20 °C). The data reveal a faster response of the transcriptome than of the metabolome and provide evidence for tightly regulated temporal responses at both levels. Metabolically, deacclimation is associated with decreasing contents of sugars, amino acids, glycolytic and TCA cycle intermediates, indicating an increased need for carbon sources and respiratory energy production for the activation of growth. The early phase of deacclimation also involves extensive down-regulation of protein synthesis and changes in the metabolism of lipids and cell wall components. Hormonal regulation appears particularly important during deacclimation, with extensive changes in the expression of genes related to auxin, gibberellin, brassinosteroid, jasmonate and ethylene metabolism. Members of several transcription factor families that control fundamental aspects of morphogenesis and development are significantly regulated during deacclimation, emphasizing that loss of freezing tolerance and growth resumption are transcriptionally highly interrelated processes. Expression patterns of some clock oscillator components resembled those under warm conditions, indicating at least partial re-activation of the circadian clock during deacclimation. CONCLUSIONS: This study provides the first combined metabolomic and transcriptomic analysis of the regulation of deacclimation in cold acclimated plants. The data indicate cascades of rapidly regulated genes and metabolites that underlie the developmental switch resulting in reduced freezing tolerance and the resumption of growth. They constitute a large-scale dataset of genes, metabolites and pathways that are crucial during the initial phase of deacclimation. The data will be an important reference for further analyses of this and other important but under-researched stress deacclimation processes.

Profiling methods to identify cold-regulated primary metabolites using gas chromatography coupled to mass spectrometry.

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of single leaves from plants that were exposed to temperature stress experiments. The described method is modular. The modules include a rapid sampling and metabolic inactivation protocol for samples in a wide size range, a sample extraction procedure, a chemical derivatization step that is required to make the metabolite fraction amenable to gas chromatographic analysis, a routine GC-MS method, and finally the procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The method has two applications: (1) the rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification of cold-regulated metabolites by exact quantification using a GC-MS protocol with extended internal and external standardization.

Alterations in cytosolic glucose-phosphate metabolism affect structural features and biochemical properties of starch-related heteroglycans.

The cytosolic pools of glucose-1-phosphate (Glc-1-P) and glucose-6-phosphate are essential intermediates in several biosynthetic paths, including the formation of sucrose and cell wall constituents, and they are also linked to the cytosolic starch-related heteroglycans. In this work, structural features and biochemical properties of starch-related heteroglycans were analyzed as affected by the cytosolic glucose monophosphate metabolism using both source and sink organs from wild-type and various transgenic potato (Solanum tuberosum) plants. In leaves, increased levels of the cytosolic phosphoglucomutase (cPGM) did affect the cytosolic heteroglycans, as both the glucosyl content and the size distribution were diminished. By contrast, underexpression of cPGM resulted in an unchanged size distribution and an unaltered or even increased glucosyl content of the heteroglycans. Heteroglycans prepared from potato tubers were found to be similar to those from leaves but were not significantly affected by the level of cPGM activity. However, external glucose or Glc-1-P exerted entirely different effects on the cytosolic heteroglycans when added to tuber discs. Glucose was directed mainly toward starch and cell wall material, but incorporation into the constituents of the cytosolic heteroglycans was very low and roughly reflected the relative monomeric abundance. By contrast, Glc-1-P was selectively taken up by the tuber discs and resulted in a fast increase in the glucosyl content of the heteroglycans that quantitatively reflected the level of the cytosolic phosphorylase activity. Based on (14)C labeling experiments, we propose that in the cytosol, glucose and Glc-1-P are metabolized by largely separated paths.