Analysis of metabolic flux using dynamic labeling and metabolic modeling

Metabolic fluxes and the capacity to modulate them are a crucial component of the ability of the plant cell to react to environmental perturbations. Our ability to quantify them and to attain information concerning the regulatory mechanisms which control them is therefore essential to understand and influence metabolic networks. For all but the simplest of flux measurements labelling methods have proven to be the most informative. Both steady-state and dynamic labelling approaches having been adopted in the study of plant metabolism. Here the conceptual basis of these complementary approaches, as well as their historical application in microbial, mammalian and plant sciences are reviewed and an update on technical developments in label distribution analyses is provided. This is supported by illustrative cases studies involving the kinetic modelling of secondary metabolism. One issue that is particularly complex in the analysis of plant fluxes is the extensive compartmentation of the plant cell. This problem is discussed from both theoretical and experimental perspectives and the current approaches used to address it are assessed. Finally, current limitations and future perspectives of kinetic modelling of plant metabolism are discussed

Integrative molecular profiling indicates a central role of transitory starch breakdown in establishing a stable C/N homeostasis during cold acclimation in two natural accessions of Arabidopsis thaliana

Figure S1. Comparison of metabolite levels between non-acclimated and acclimated plants. Ratios were built by dividing the absolute mean values of metabolite levels of Rsch by levels of Cvi, or by dividing absolute mean values of metabolites of acc by na plants. Asterisks indicate significant differences as described in the figure. Grey-coloured metabolites were not experimentally analysed. (TIF 1649 kb

Label-free proteomic analysis of Xerophyta schlechteri leaf tissue under dehydration stress

Most higher plants cannot withstand severe water loss, except for a small group of angiosperms called resurrection plants. They can survive severe water loss without the loss of viability by employing mechanisms that aid them in desiccation tolerance. Desiccation tolerance in resurrection plants is a complex and multifaceted phenomenon and allows the plant to implement various strategies for survival. The focus of this study was a label-free proteomic analysis of Xerophyta schlechteri, a monocotyledonous and poikilochlorophyllous resurrection plant, in response to desiccation. The study investigated some of the physiological, morphological and biochemical changes of X. schlechteri leaf tissue in response to dehydration followed by proteomic analyses using a spectral counting approach. The differentially expressed proteins were identified and quantified and then subjected to gene ontological analyses to identify relevant biological processes involved in desiccation tolerance. The proteomic data was finally correlated to and validated using metabolomic analyses. X. schlechteri was subjected to a controlled dehydration stress treatment, in which changes in the relative water content (RWC) of leaf tissues, the associated changes in processes outlined above and further expanded on below, were determined. Three physiological stages were tentatively identified, namely, the early response to drying (ERD) which represents ~ 80 - 70% RWC (1.61 gH2O g ̄ˡ dwt -1.5 gH2O g ̄ˡ dwt), a mid-response to drying (MRD) represented by ~ 60 - 40% RWC (1.5 gH2O g ̄ˡ dwt -1.0 gH2O g ̄ˡ dwt) and a late response to drying (LRD), represented by ~ 40 - 10% RWC (1.0 gH2O g ̄ˡ dwt - 0.5 gH2O g ̄ˡ dwt). Morphological changes in the late stages of drying were marked by loss of green chlorophyll, increased purple anthocyanin production and leaf folding along the midrib with the abaxial surface exposed to light. Chlorophyll content analyses showed a significant decrease in chlorophyll content in the dehydrated leaf tissue as compared to the fully hydrated state. Biochemical assays to measure the activity of enzymatic antioxidants, namely, ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) were done at selected RWC points. There was a significant increase in antioxidant enzyme activity for APX, CAT, GR and SOD in the dehydrated plant tissue. The label-free proteomics approach utilized, identified a total of 3125 unique proteins in the X. schlechteri leaf tissue across the dehydration treatment of which a combined 517 proteins were significantly differentially expressed in response to drying. Amongst the differentially expressed proteins, 253 proteins were upregulated, and 264 proteins were downregulated. This was followed by functional analyses and classification of gene ontologies using bioinformatics tools such as Blast2GO, MapMan and KEGG. This allowed the identification of certain biological processes and pathways involved in the X. schlechteri desiccation response. Key biological processes and molecular processes were differentially expressed across the drying stages, these included photosynthesis, cellular respiration and antioxidant activity, respectively. The proteomic analysis was complemented and validated using metabolomics approaches based on GC MS/MS and LC/MS. The abundance of specific sugars, sugar alcohols, fatty acids, organic acids, phytohormones and amino acids of X. schlechteri during desiccation were investigated. Sugars such as raffinose and sucrose are known to play a protective role in desiccation and were found to be abundant in MRD and LRD leaf tissue while, L-histidine, an amino acid which plays a critical role in plant growth, was found to be more abundant in LRD tissue as compared to MRD. The phytohormone abscisic acid, invoked in desiccation tolerance was found to be abundant at LRD and less abundant at ERD. The metabolomic data suggested that the regulation of metabolites was towards reducing possible toxic metabolites while increasing the expression of metabolites that help and protect plant cell integrity from the negative effects of desiccation. The use of a label-free proteomics approach complemented with metabolomics allowed the identification and validation of biological processes and pathways potentially involved in establishing desiccation tolerance in X. schlechteri. As far as we are aware, this is the first label-free proteomic analysis of X. schlechteri in response to dehydration

Impaired chloroplast positioning affects photosynthetic capacity and regulation of the central carbohydrate metabolism during cold acclimation

Photosynthesis and carbohydrate metabolism of higher plants need to be tightly regulated to prevent tissue damage during environmental changes. The intracellular position of chloroplasts changes due to a changing light regime. Chloroplast avoidance and accumulation response under high and low light, respectively, are well known phenomena, and deficiency of chloroplast movement has been shown to result in photodamage and reduced biomass accumulation. Yet, effects of chloroplast positioning on underlying metabolic regulation are less well understood. Here, we analysed photosynthesis together with metabolites and enzyme activities of the central carbohydrate metabolism during cold acclimation of the chloroplast unusual positioning 1 (chup1) mutant of Arabidopsis thaliana. We compared cold acclimation under ambient and low light and found that maximum quantum yield of PSII was significantly lower in chup1 than in Col-0 under both conditions. Our findings indicated that net CO2 assimilation in chup1 is rather limited by biochemistry than by photochemistry. Further, cold-induced dynamics of sucrose phosphate synthase differed significantly between both genotypes. Together with a reduced rate of sucrose cycling derived from kinetic model simulations our study provides evidence for a central role of chloroplast positioning for photosynthetic and metabolic acclimation to low temperature

Transcriptome analysis by GeneTrail revealed regulation of functional categories in response to alterations of iron homeostasis in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>High-throughput technologies have opened new avenues to study biological processes and pathways. The interpretation of the immense amount of data sets generated nowadays needs to be facilitated in order to enable biologists to identify complex gene networks and functional pathways. To cope with this task multiple computer-based programs have been developed. GeneTrail is a freely available online tool that screens comparative transcriptomic data for differentially regulated functional categories and biological pathways extracted from common data bases like KEGG, Gene Ontology (GO), TRANSPATH and TRANSFAC. Additionally, GeneTrail offers a feature that allows screening of individually defined biological categories that are relevant for the respective research topic.</p> <p>Results</p> <p>We have set up GeneTrail for the use of <it>Arabidopsis thaliana</it>. To test the functionality of this tool for plant analysis, we generated transcriptome data of root and leaf responses to Fe deficiency and the Arabidopsis metal homeostasis mutant <it>nas4x-1</it>. We performed Gene Set Enrichment Analysis (GSEA) with eight meaningful pairwise comparisons of transcriptome data sets. We were able to uncover several functional pathways including metal homeostasis that were affected in our experimental situations. Representation of the differentially regulated functional categories in Venn diagrams uncovered regulatory networks at the level of whole functional pathways. Over-Representation Analysis (ORA) of differentially regulated genes identified in pairwise comparisons revealed specific functional plant physiological categories as major targets upon Fe deficiency and in <it>nas4x-1</it>.</p> <p>Conclusion</p> <p>Here, we obtained supporting evidence, that the <it>nas4x-1 </it>mutant was defective in metal homeostasis. It was confirmed that <it>nas4x-1 </it>showed Fe deficiency in roots and signs of Fe deficiency and Fe sufficiency in leaves. Besides metal homeostasis, biotic stress, root carbohydrate, leaf photosystem and specific cell biological categories were discovered as main targets for regulated changes in response to - Fe and <it>nas4x-1</it>. Among 258 differentially expressed genes in response to - Fe and <it>nas4x-1 </it>five functional categories were enriched covering metal homeostasis, redox regulation, cell division and histone acetylation. We proved that GeneTrail offers a flexible and user-adapted way to identify functional categories in large-scale plant transcriptome data sets. The distinguished feature that allowed analysis of individually assembled functional categories facilitated the study of the <it>Arabidopsis thaliana </it>transcriptome.</p

Predicting plant growth response under fluctuating temperature by carbon balance modelling

Quantification of system dynamics is a central aim of mathematical modelling in biology. Defining experimentally supported functional relationships between molecular entities by mathematical terms enables the application of computational routines to simulate and analyse the underlying molecular system. In many fields of natural sciences and engineering, trigonometric functions are applied to describe oscillatory processes. As biochemical oscillations occur in many aspects of biochemistry and biophysics, Fourier analysis of metabolic functions promises to quantify, describe and analyse metabolism and its reaction towards environmental fluctuations. Here, Fourier polynomials were developed from experimental time-series data and combined with block diagram simulation of plant metabolism to study heat shock response of photosynthetic CO 2 assimilation and carbohydrate metabolism in Arabidopsis thaliana . Simulations predicted a stabilising effect of reduced sucrose biosynthesis capacity and increased capacity of starch biosynthesis on carbon assimilation under transient heat stress. Model predictions were experimentally validated by quantifying plant growth under such stress conditions. In conclusion, this suggests that Fourier polynomials represent a predictive mathematical approach to study dynamic plant-environment interactions

Metabolic interactions between Plasmodiophora brassicae and Arabidopsis thaliana plant

Clubroot (Plasmodiophora brassicae) is a serious agricultural problem affecting Brassica crops. It also infects Arabidopsis thaliana plants. During infection, this biotrophic pathogen manipulates the development and metabolism of its host leading to the development of galls. In turn, its own development is strongly influenced by the host. The aim of this study was to understand the metabolic interaction between A. thaliana plants and P. brassicae. An initial non-targeted approach was used to obtain metabolic `fingerprints’, which were then combined with host transcriptomic data. In addition, a targeted approach was applied focusing on carbohydrate metabolism. Hypotheses were identified using transcriptomic data and tested using mutants of A. thaliana and analysis of reporter gene expression. Changes in plant development occurring as a consequence of clubroot infection correlated with changes in metabolic status were investigated. Following P. brassicae infection, metabolite profiles altered at the beginning of cortical infection, although plant primary growth did not show clear differences between uninfected and infected tissue at this stage. This suggests that these changes in metabolites depended on responses of the plant to infection rather changes in plant development. The accumulation of the amino acids glutamate, aspartate and alanine are likely to be related to pathogen nutrition. Metabolites such as proline protect plants from osmotic and oxidative stress. Meanwhile, compounds associated with plant defence such as cinnamic acid and phaseic acid accumulated at 16 DPI and decreased at 26 DPI. The accumulation of vitamin B6 precursor and compounds associated with folate biosynthesis were accompanied with increasing host gene expression associated with the synthesis of these metabolites. The accumulation of other metabolites such as thiosulfate was accompanied with the repression of genes associated with their degradation. This suggests that P. brassicae has the potential to suppress the expression of host metabolism genes to obtain nutrients from the host. Transcriptomic analysis showed that sucrose synthase (SUS) and sugar permeases were induced during gall formation. The impact of inactivating these genes (and cytosolic invertase CINV) on gall formation was examined. In wildtype plants the hypocotyl width was not affected at 16 DPI, but increased by 26 DPI. Similar results were seen in cinv1,2 and sus1-,4 plants at 16 DPI. By 26 DPI, cinv1,2 and sus1-4 plants showed a smaller hypocotyl width than Col-0 plants when uninfected, but this difference was not evident in sus1-4 plants when infected. Infected cinv1,2 plants were smaller than Col-0 plants at 26 DPI, although plasmodia colonized host cells and pathogen development was similar to that in Col-0 plants. This indicates that P. brassicae itself makes the gall a sink. Meanwhile, sweet11,12 mutants displayed slower P. brassicae development due to a change in carbohydrate partitioning. SWEET::GUS expression patterns support the hypothesis that sucrose was transported to plasmodia via these transporters

Organellar carbon metabolism is co-ordinated with distinct developmental phases of secondary xylem

Subcellular compartmentation of plant biosynthetic pathways in the mitochondria and plastids requires coordinated regulation of nuclear encoded genes, and the role of these genes has been largely ignored by wood researchers. In this study, we constructed a targeted systems genetics coexpression network of xylogenesis in Eucalyptus using plastid and mitochondrial carbon metabolic genes and compared the resulting clusters to the aspen xylem developmental series. The constructed network clusters reveal the organization of transcriptional modules regulating subcellular metabolic functions in plastids and mitochondria. Overlapping genes between the plastid and mitochondrial networks implicate the common transcriptional regulation of carbon metabolism during xylem secondary growth. We show that the central processes of organellar carbon metabolism are distinctly coordinated across the developmental stages of wood formation and are specifically associated with primary growth and secondary cell wall deposition. We also demonstrate that, during xylogenesis, plastid-targeted carbon metabolism is partially regulated by the central clock for carbon allocation towards primary and secondary xylem growth, and we discuss these networks in the context of previously established associations with wood-related complex traits. This study provides a new resolution into the integration and transcriptional regulation of plastid- and mitochondrial-localized carbon metabolism during xylogenesis

Responses of starch potatoes (Solanum tuberosum L.) to osmotic stress in vitro regarding growth, proteome and metabolite profile

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Metabolic profiling of Arabidopsis thaliana epidermal cells

Metabolic phenotyping at cellular resolution may be considered one of the challenges in current plant physiology. A method is described which enables the cell type-specific metabolic analysis of epidermal cell types in Arabidopsis thaliana pavement, basal, and trichome cells. To achieve the required high spatial resolution, single cell sampling using microcapillaries was combined with routine gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) based metabolite profiling. The identification and relative quantification of 117 mostly primary metabolites has been demonstrated. The majority, namely 90 compounds, were accessible without analytical background correction. Analyses were performed using cell type-specific pools of 200 microsampled individual cells. Moreover, among these identified metabolites, 38 exhibited differential pool sizes in trichomes, basal or pavement cells. The application of an independent component analysis confirmed the cell type-specific metabolic phenotypes. Significant pool size changes between individual cells were detectable within several classes of metabolites, namely amino acids, fatty acids and alcohols, alkanes, lipids, N-compounds, organic acids and polyhydroxy acids, polyols, sugars, sugar conjugates and phenylpropanoids. It is demonstrated here that the combination of microsampling and GC-MS based metabolite profiling provides a method to investigate the cellular metabolism of fully differentiated plant cell types in vivo