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Plant growth occurs via the interconnection of cell growth and proliferation in each organ following specific developmental and environmental cues. Therefore, different photoperiods result in distinct growth patterns due to the integration of light and circadian perception with specific Carbon (C) partitioning strategies. In addition, the TARGET OF RAPAMYCIN (TOR) kinase pathway is an ancestral signaling pathway that integrates nutrient information with translational control and growth regulation. Recent findings in Arabidopsis (Arabidopsis thaliana) have shown a mutual connection between the TOR pathway and the circadian clock. However, the mechanistical network underlying this interaction is mostly unknown. Here, we show that the conserved TOR target, the 40S ribosomal protein S6 kinase (S6K) is under circadian and photoperiod regulation both at the transcriptional and post-translational level. Total S6K (S6K1 and S6K2) and TOR-dependent phosphorylated-S6K protein levels were higher during the light period and decreased at dusk especially under short day conditions. Using chemical and genetic approaches, we found that the diel pattern of S6K accumulation results from 26S proteasome-dependent degradation and is altered in mutants lacking the circadian F-box protein ZEITLUPE (ZTL), further strengthening our hypothesis that S6K could incorporate metabolic signals via TOR, which are also under circadian regulation. Moreover, under short days when C/energy levels are limiting, changes in S6K1 protein levels affected starch, sucrose and glucose accumulation and consequently impacted root and rosette growth responses. In summary, we propose that S6K1 constitutes a missing molecular link where day-length perception, nutrient availability and TOR pathway activity converge to coordinate growth responses with environmental conditions., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Carbon (C) and nitrogen (N) metabolisms are tightly integrated to allow proper plant growth and development. Photosynthesis is dependent on N invested in chlorophylls, enzymes, and structural components of the photosynthetic machinery, while N uptake and assimilation rely on ATP, reducing equivalents, and C-skeletons provided by photosynthesis. The direct connection between N availability and photosynthetic efficiency allows the synthesis of precursors for all metabolites and building blocks in plants. Thus, the capacity to sense and respond to sudden changes in C and N availability is crucial for plant survival and is mediated by complex yet efficient signaling pathways such as TARGET OF RAPAMYCIN (TOR) and SUCROSE-NON-FERMENTING-1-RELATED PROTEIN KINASE 1 (SnRK1). In this review, we present recent advances in mechanisms involved in sensing C and N status as well as identifying current gaps in our understanding. We finally attempt to provide new perspectives and hypotheses on the interconnection of diverse signaling pathways that will allow us to understand the integration and orchestration of the major players governing the regulation of the CN balance., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant growth is intimately linked to the availability of carbon and energy status. The Target of rapamycin (TOR) pathway is a highly relevant metabolic sensor and integrator of plant-assimilated C into development and growth. The cell wall accounts for around a third of the cell biomass, and the investment of C into this structure should be finely tuned for optimal growth. The plant C status plays a significant role in controlling the rate of cell wall synthesis. TOR signaling regulates cell growth and expansion, which are fundamental processes for plant development. The availability of nutrients and energy, sensed and integrated by TOR, influences cell division and elongation, ultimately impacting the synthesis and deposition of cell wall components. The plant cell wall is crucial in environmental adaptation and stress responses. TOR senses and internalizes various environmental cues, such as nutrient availability and stresses. These environmental factors influence TOR activity, which modulates cell wall remodeling to cope with changing conditions. Plant hormones, including auxins, gibberellins, and brassinosteroids, also regulate TOR signaling and cell wall-related processes. The connection between nutrients and cell wall pathways modulated by TOR are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Starch serves as an important carbon storage mechanism for many plant species, facilitating their adaptation to the cyclic variations in the light environment, including day-night cycles as well as seasonal changes in photoperiod. By dynamically adjusting starch accumulation and degradation rates, plants maintain carbon homeostasis, enabling continuous growth under fluctuating environmental conditions. To understand dynamic nature of starch metabolism at the molecular level, it is necessary to integrate empirical knowledge from genetic defects in specific regulatory pathways into the dynamical system of starch metabolism. To achieve this, we evaluated the impact of genetic defects in the circadian clock, sugar sensing and starch degradation pathways using the carbon homeostasis model that encompasses the interplay between these pathways. Through the collection of starch metabolism data from 10 Arabidopsis mutants, we effectively fitted the experimental data to the model. The system-level assessment revealed that genetic defects in both circadian clock components and sugar sensing pathway hindered the appropriate adjustment of the starch degradation rate, particularly under long-day conditions. These findings not only confirmed the previous empirical findings but also provide the novel insights into the role of each gene within the gene regulatory network on the emergence of carbon homeostasis.

Developmental transitions, occurring throughout the life cycle of plants, require precise regulation of metabolic processes to generate the energy and resources necessary for the committed growth processes. In parallel, the establishment of new cells, tissues, and even organs, alongside their differentiation provoke profound changes in metabolism. It is increasingly being recognized that there is a certain degree of feedback regulation between the components and products of metabolic pathways and developmental regulators. The generation of large-scale metabolomics datasets during developmental transitions, in combination with molecular genetic approaches has helped to further our knowledge on the functional importance of metabolic regulation of development. In this perspective article, we provide insights into studies that elucidate interactions between metabolism and development at the temporal and spatial scales. We additionally discuss how this influences cell growth-related processes. We also highlight how metabolic intermediates function as signaling molecules to direct plant development in response to changing internal and external conditions., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

As a comprehensive analysis of all metabolites in a biological system, metabolomics is being widely applied in various clinical/health areas for disease prediction, diagnosis, and prognosis. However, challenges remain in dealing with the metabolomic complexity, massive data, metabolite identification, intra- and inter-individual variation, and reproducibility, which largely limit its widespread implementation. This study provided a comprehensive workflow for clinical metabolomics, including sample collection and preparation, mass spectrometry (MS) data acquisition, and data processing and analysis. Sample collection from multiple clinical sites was strictly carried out with standardized operation procedures (SOP). During data acquisition, three types of quality control (QC) samples were set for respective MS platforms (GC-MS, LC-MS polar, and LC-MS lipid) to assess the MS performance, facilitate metabolite identification, and eliminate contamination. Compounds annotation and identification were implemented with commercial software and in-house-developed PAppLine TM and Ulib MS library. The batch effects were removed using a deep learning model method (NormAE). Potential biomarkers identification was performed with tree-based modeling algorithms including random forest, AdaBoost, and XGBoost. The modeling performance was evaluated using the F1 score based on a 10-times repeated trial for each. Finally, a sub-cohort case study validated the reliability of the entire workflow.

While metabolism has been recognized as a key regulator of plant development, exactly how this is achieved is unknown. A new study identifies a component of the Polycomb repressor complex 2 as linking these processes via histone modification., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Plants can adjust their growth to specific times of the day and season. Different photoperiods result in distinct growth patterns, which correlate with specific carbon-partitioning strategies in source (leaves) and sink (roots) organs. Therefore, external cues such as light, day length, and temperature need to be integrated with intracellular processes controlling overall carbon availability and anabolism. The target of rapamycin (TOR) pathway is a signalling hub where environmental signals, circadian information, and metabolic processes converge to regulate plant growth. TOR complex mutants display altered patterns of root growth and starch levels. Moreover, depletion of TOR or reduction in cellular energy levels affect the pace of the clock by extending the period length, suggesting that this pathway could participate in circadian metabolic entrainment. However, this seems to be a mutual interaction, since the TOR pathway components are also under circadian regulation. These results strengthen the role of this signalling pathway as a master sensor of metabolic status, integrating day length and circadian cues to control anabolic processes in the cell, thus promoting plant growth and development. Expanding this knowledge from Arabidopsis thaliana to crops will improve our understanding of the molecular links connecting environmental perception and growth regulation under field conditions., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Sugars are essential metabolites for energy and anabolism that can also act as signals to regulate plant physiology and development. Experimental tools to disrupt major sugar signalling pathways are limited. We performed a chemical screen for modifiers of activation of circadian gene expression by sugars to discover pharmacological tools to investigate and manipulate plant sugar signalling. Using a library of commercially available bioactive compounds, we identified 75 confident hits that modified the response of a circadian luciferase reporter to sucrose in dark-adapted Arabidopsis thaliana seedlings. We validated the transcriptional effect on a subset of the hits and measured their effects on a range of sugar-dependent phenotypes for 13 of these chemicals. Chemicals were identified that appear to influence known and unknown sugar signalling pathways. Pentamidine isethionate was identified as a modifier of a sugar-activated Ca 2+ signal that acts as a calmodulin inhibitor downstream of superoxide in a metabolic signalling pathway affecting circadian rhythms, primary metabolism and plant growth. Our data provide a resource of new experimental tools to manipulate plant sugar signalling and identify novel components of these pathways., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Phosphorus (P) is a vital nutrient for plant growth. P availability is generally low in soils, and plant responses to low P availability need to be better understood. In a previous study, we studied the growth and physiological responses of 24 species to low P availability in the soil and verified of eucalypts, five ( Eucalyptus acmenoides , E. grandis , E. globulus , E. tereticornis , and Corymbia maculata ) contrasted regarding their efficiency and responsiveness to soil P availability. Here, we obtained the metabolomic and lipidomic profile of leaves, stems, and roots from these species growing under low (4.5 mg dm -3 ) and sufficient (10.8 mg dm -3 ) P in the soil. Disregarding the level of P in the soils, P allocation was always higher in the stems. However, when grown in the P-sufficient soil, the stems steadily were the largest compartment of the total plant P. Under low P, the relative contents of primary metabolites, such as amino acids, TCA cycle intermediates, organic acids and carbohydrates, changed differently depending on the species. Additionally, phosphorylated metabolites showed enhanced turnover or reductions. While photosynthetic efficiencies were not related to higher biomass production, A / C i curves showed that reduced P availability increased the eucalypt species' Vcmax, Jmax and photosynthetic P-use efficiency . Plants of E. acmenoides increased galactolipids and sulfolipids in leaves more than other eucalypt species, suggesting that lipid remodelling can be a strategy to cope with the P shortage in this species. Our findings offer insights to understand genotypic efficiency among eucalypt species to accommodate primary metabolism under low soil P availability and eventually be used as biochemical markers for breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Silva, Bulgarelli, Mubeen, Caldana, Andrade and Mazzafera.)

Witches' broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plants, as autotrophic organisms, capture light energy to convert carbon dioxide into ATP, NADPH, and sugars, which are essential for the biosynthesis of building blocks, cell proliferation, biomass accumulation, and reproductive fitness. The Target Of Rapamycin (TOR) signalling pathway is a master regulator in sensing energy and nutrients, adapting the metabolic network and cell behaviour in response to environmental resource availability. In the past years, exciting advances in this endeavour have pointed out this pathway's importance in controlling metabolic homeostasis in various biological processes and systems. In this review, we discuss these recent discoveries highlighting the need for a metabolic threshold for the proper function of this kinase complex at the cellular level and across distinct tissues and organs to control growth and development in plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As autotrophic organisms, plants capture light energy to convert carbon dioxide into ATP, nicotinamide adenine dinucleotide phosphate (NADPH), and sugars, which are essential for the biosynthesis of building blocks, storage, and growth. At night, metabolism and growth can be sustained by mobilizing carbon (C) reserves. In response to changing environmental conditions, such as light-dark cycles, the small-molecule regulation of enzymatic activities is critical for reprogramming cellular metabolism. We have recently demonstrated that proteogenic dipeptides, protein degradation products, act as metabolic switches at the interface of proteostasis and central metabolism in both plants and yeast. Dipeptides accumulate in response to the environmental changes and act via direct binding and regulation of critical enzymatic activities, enabling C flux distribution. Here, we provide evidence pointing to the involvement of dipeptides in the metabolic rewiring characteristics for the day-night cycle in plants. Specifically, we measured the abundance of 13 amino acids and 179 dipeptides over short- (SD) and long-day (LD) diel cycles, each with different light intensities. Of the measured dipeptides, 38 and eight were characterized by day-night oscillation in SD and LD, respectively, reaching maximum accumulation at the end of the day and then gradually falling in the night. Not only the number of dipeptides, but also the amplitude of the oscillation was higher in SD compared with LD conditions. Notably, rhythmic dipeptides were enriched in the glucogenic amino acids that can be converted into glucose. Considering the known role of Target of Rapamycin (TOR) signaling in regulating both autophagy and metabolism, we subsequently investigated whether diurnal fluctuations of dipeptides levels are dependent on the TOR Complex (TORC). The Raptor1b mutant ( raptor1b ), known for the substantial reduction of TOR kinase activity, was characterized by the augmented accumulation of dipeptides, which is especially pronounced under LD conditions. We were particularly intrigued by the group of 16 dipeptides, which, based on their oscillation under SD conditions and accumulation in raptor1b , can be associated with limited C availability or photoperiod. By mining existing protein-metabolite interaction data, we delineated putative protein interactors for a representative dipeptide Pro-Gln. The obtained list included enzymes of C and amino acid metabolism, which are also linked to the TORC-mediated metabolic network. Based on the obtained results, we speculate that the diurnal accumulation of dipeptides contributes to its metabolic adaptation in response to changes in C availability. We hypothesize that dipeptides would act as alternative respiratory substrates and by directly modulating the activity of the focal enzymes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Calderan-Rodrigues, Luzarowski, Monte-Bello, Minen, Zühlke, Nikoloski, Skirycz and Caldana.)

Key Message: The tomato mutant Never ripe (Nr), a loss-of-function for the ethylene receptor SlETR3, shows enhanced growth, associated with increased carbon assimilation and a rewiring of the central metabolism. Compelling evidence has demonstrated the importance of ethylene during tomato fruit development, yet its role on leaf central metabolism and plant growth remains elusive. Here, we performed a detailed characterization of Never ripe (Nr) tomato, a loss-of-function mutant for the ethylene receptor SlETR3, known for its fruits which never ripe. However, besides fruits, the Nr gene is also constitutively expressed in vegetative tissues. Nr mutant showed a growth enhancement during both the vegetative and reproductive stage, without an earlier onset of leaf senescence, with Nr plants exhibiting a higher number of leaves and an increased dry weight of leaves, stems, roots, and fruits. At metabolic level, Nr also plays a significant role with the mutant showing changes in carbon assimilation, carbohydrates turnover, and an exquisite reprogramming of a large number of metabolite levels. Notably, the expression of genes related to ethylene signaling and biosynthesis are not altered in Nr. We assess our results in the context of those previously published for tomato fruits and of current models of ethylene signal transduction, and conclude that ethylene insensitivity mediated by Nr impacts the whole central metabolism at vegetative stage, leading to increased growth rates., (© 2020. Springer-Verlag GmbH Germany, part of Springer Nature.)

N-Acetylcysteine (NAC) is an antioxidant, anti-adhesive, and antimicrobial compound. Even though there is much information regarding the role of NAC as an antioxidant and anti-adhesive agent, little is known about its antimicrobial activity. In order to assess its mode of action in bacterial cells, we investigated the metabolic responses triggered by NAC at neutral pH. As a model organism, we chose the Gram-negative plant pathogen Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker disease, due to the potential use of NAC as a sustainable molecule against phytopathogens dissemination in citrus cultivated areas. In presence of NAC, cell proliferation was affected after 4 h, but damages to the cell membrane were observed only after 24 h. Targeted metabolite profiling analysis using GC-MS/TOF unravelled that NAC seems to be metabolized by the cells affecting cysteine metabolism. Intriguingly, glutamine, a marker for nitrogen status, was not detected among the cells treated with NAC. The absence of glutamine was followed by a decrease in the levels of the majority of the proteinogenic amino acids, suggesting that the reduced availability of amino acids affect protein synthesis and consequently cell proliferation., (© 2021. The Author(s).)

The Target of Rapamycin (TOR) kinase pathway integrates energy and nutrient availability into metabolism promoting growth in eukaryotes. The overall higher efficiency on nutrient use translated into faster growth rates in C 4 grass plants led to the investigation of differential transcriptional and metabolic responses to short-term chemical TOR complex (TORC) suppression in the model Setaria viridis . In addition to previously described responses to TORC inhibition (i.e., general growth arrest, translational repression, and primary metabolism reprogramming) in Arabidopsis thaliana (C 3 ), the magnitude of changes was smaller in S. viridis , particularly regarding nutrient use efficiency and C allocation and partitioning that promote biosynthetic growth. Besides photosynthetic differences, S. viridis and A. thaliana present several specificities that classify them into distinct lineages, which also contribute to the observed alterations mediated by TOR. Indeed, cell wall metabolism seems to be distinctly regulated according to each cell wall type, as synthesis of non-pectic polysaccharides were affected in S. viridis , whilst assembly and structure in A. thaliana. Our results indicate that the metabolic network needed to achieve faster growth seems to be less stringently controlled by TORC in S. viridis ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 da Silva, Martins, Calderan-Rodrigues, Artins, Monte Bello, Gupta, Sobreira, Riaño-Pachón, Mafra and Caldana.)

Metabolomics is a valuable approach used to acquire comprehensive information about the set of metabolites in a cell or tissue, enabling a functional screen of the cellular activities in biological systems. Although metabolomics provides a more immediate and dynamic picture of phenotypes in comparison to the other omics, it is also the most complicated to measure because no single analytical technology can capture the extraordinary complexity of metabolite diversity in terms of structure and physical properties. Metabolomics has been extensively employed for a wide range of applications in plant science, which will be described in detail in this chapter. Among them, metabolomics is used for discriminating patterns of plant responses to genetic and environmental perturbations, as diagnostics and prediction tool to elucidate the function of genes for important and complex agronomic traits in crop species, and flux measurements are used to dissect the structure and regulatory properties of metabolic networks., (© 2021. Springer Nature Switzerland AG.)

The clockwork of plant circadian oscillators has been resolved through investigations in Arabidopsis thaliana. The circadian oscillator is an important regulator of much of plant physiology, though many of the mechanisms are unclear. New findings demonstrate that the oscillator adjusts phase and period in response to abiotic and biotic signals, providing insight in to how the plant circadian oscillator integrates with the biology of the cell and entrains to light, dark and temperature cycles. We propose that the plant circadian oscillator is dynamically plastic, in constant adjustment, rather than being an isolated clock impervious to cellular events.

The increase in atmospheric CO 2 due to anthropogenic activities is generating climate change, which has resulted in a subsequent rise in global temperatures with severe environmental impacts. Biological mitigation has been considered as an alternative for environmental remediation and reduction of greenhouse gases in the atmosphere. In fact, the use of easily adapted photosynthetic organisms able to fix CO 2 with low-cost operation is revealing its high potential for industry. Among those organism, the algae Chlamydomonas reinhardtii have gain special attention as a model organism for studying CO 2 fixation, biomass accumulation and bioenergy production upon exposure to several environmental conditions. In the present study, we studied the Chlamydomonas response to different CO 2 levels by comparing metabolomics and transcriptomics data with the predicted results from our new-improved genomic-scale metabolic model. For this, we used in silico methods at steady dynamic state varying the levels of CO 2 . Our main goal was to improve our capacity for predicting metabolic routes involved in biomass accumulation. The improved genomic-scale metabolic model presented in this study was shown to be phenotypically accurate, predictive, and a significant improvement over previously reported models. Our model consists of 3726 reactions and 2436 metabolites, and lacks any thermodynamically infeasible cycles. It was shown to be highly sensitive to environmental changes under both steady-state and dynamic conditions. As additional constraints, our dynamic model involved kinetic parameters associated with substrate consumption at different growth conditions (i.e., low CO 2 -heterotrophic and high CO 2 -mixotrophic). Our results suggest that cells growing at high CO 2 (i.e., photoautotrophic and mixotrophic conditions) have an increased capability for biomass production. In addition, we have observed that ATP production also seems to be an important limiting factor for growth under the conditions tested. Our experimental data (metabolomics and transcriptomics) and the results predicted by our model clearly suggest a differential behavior between low CO 2 -heterotrophic and high CO 2 -mixotrophic growth conditions. The data presented in the current study contributes to better dissect the biological response of C. reinhardtii, as a dynamic entity, to environmental and genetic changes. These findings are of great interest given the biotechnological potential of this microalga for CO 2 fixation, biomass accumulation, and bioenergy production., Competing Interests: The authors declare there are no competing interests.

The pyruvate dehydrogenase complex (PDH), that converts pyruvate to acetyl-coA, is regulated by pyruvate dehydrogenase kinases (PDHK) and phosphatases (PDHP) that have been shown to be important for morphology, pathogenicity and carbon source utilization in different fungal species. The aim of this study was to investigate the role played by the three PDHKs PkpA, PkpB and PkpC in carbon source utilization in the reference filamentous fungus Aspergillus nidulans , in order to unravel regulatory mechanisms which could prove useful for fungal biotechnological and biomedical applications. PkpA and PkpB were shown to be mitochondrial whereas PkpC localized to the mitochondria in a carbon source-dependent manner. Only PkpA was shown to regulate PDH activity. In the presence of glucose, deletion of pkpA and pkpC resulted in reduced glucose utilization, which affected carbon catabolite repression (CCR) and hydrolytic enzyme secretion, due to de-regulated glycolysis and TCA cycle enzyme activities. Furthermore, PkpC was shown to be required for the correct metabolic utilization of cellulose and acetate. PkpC negatively regulated the activity of the glyoxylate cycle enzyme isocitrate lyase (ICL), required for acetate metabolism. In summary, this study identified PDHKs important for the regulation of central carbon metabolism in the presence of different carbon sources, with effects on the secretion of biotechnologically important enzymes and carbon source-related growth. This work demonstrates how central carbon metabolism can affect a variety of fungal traits and lays a basis for further investigation into these characteristics with potential interest for different applications., (Copyright © 2018 Ries et al.)

Background: Lignin is a heterogeneous polymer representing a renewable source of aromatic and phenolic bio-derived products for the chemical industry. However, the inherent structural complexity and recalcitrance of lignin makes its conversion into valuable chemicals a challenge. Natural microbial communities produce biocatalysts derived from a large number of microorganisms, including those considered unculturable, which operate synergistically to perform a variety of bioconversion processes. Thus, metagenomic approaches are a powerful tool to reveal novel optimized metabolic pathways for lignin conversion and valorization., Results: The lignin-degrading consortium (LigMet) was obtained from a sugarcane plantation soil sample. The LigMet taxonomical analyses (based on 16S rRNA) indicated prevalence of Proteobacteria , Actinobacteria and Firmicutes members, including the Alcaligenaceae and Micrococcaceae families, which were enriched in the LigMet compared to sugarcane soil. Analysis of global DNA sequencing revealed around 240,000 gene models, and 65 draft bacterial genomes were predicted. Along with depicting several peroxidases, dye-decolorizing peroxidases, laccases, carbohydrate esterases, and lignocellulosic auxiliary (redox) activities, the major pathways related to aromatic degradation were identified, including benzoate (or methylbenzoate) degradation to catechol (or methylcatechol), catechol ortho-cleavage, catechol meta-cleavage, and phthalate degradation. A novel Paenarthrobacter strain harboring eight gene clusters related to aromatic degradation was isolated from LigMet and was able to grow on lignin as major carbon source. Furthermore, a recombinant pathway for vanillin production was designed based on novel gene sequences coding for a feruloyl-CoA synthetase and an enoyl-CoA hydratase/aldolase retrieved from the metagenomic data set., Conclusion: The enrichment protocol described in the present study was successful for a microbial consortium establishment towards the lignin and aromatic metabolism, providing pathways and enzyme sets for synthetic biology engineering approaches. This work represents a pioneering study on lignin conversion and valorization strategies based on metagenomics, revealing several novel lignin conversion enzymes, aromatic-degrading bacterial genomes, and a novel bacterial strain of potential biotechnological interest. The validation of a biosynthetic route for vanillin synthesis confirmed the applicability of the targeted metagenome discovery approach for lignin valorization strategies.

The output of metabolomics relies to a great extent upon the methods and instrumentation to identify, quantify, and access spatial information on as many metabolites as possible. However, the most modern machines and sophisticated tools for data analysis cannot compensate for inappropriate harvesting and/or sample preparation procedures that modify metabolic composition and can lead to erroneous interpretation of results. In addition, plant metabolism has a remarkable degree of complexity, and the number of identified compounds easily surpasses the number of samples in metabolomics analyses, increasing false discovery risk. These aspects pose a large challenge when carrying out plant metabolomics experiments. In this chapter, we address the importance of a proper experimental design taking into consideration preventable complications and unavoidable factors to achieve success in metabolomics analysis. We also focus on quality control and standardized procedures during the metabolomics workflow.

Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15 million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus., Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli., Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways., (© 2016 John Wiley & Sons Ltd.)

A number of Arabidopsis (Arabidopsis thaliana) mutants exhibit leaf reticulation, having green veins that stand out against paler interveinal tissues, fewer cells in the interveinal mesophyll, and normal perivascular bundle sheath cells. Here, to examine the basis of leaf reticulation, we analyzed the Arabidopsis RETICULATA-RELATED (RER) gene family, several members of which cause leaf reticulation when mutated. Although transcripts of RE, RER1, and RER3 were mainly detected in the bundle sheath cells of expanded leaves, functional RER3:GREEN FLUORESCENT PROTEIN was visualized in the chloroplast membranes of all photosynthetic cells. Leaf reticulation in the re and rer3 loss-of-function mutants occurred, along with accumulation of reactive oxygen species, in a photoperiod-dependent manner. A comparison of re and rer3 leaf messenger RNA expression profiles showed more than 200 genes were similarly misexpressed in both mutants. In addition, metabolic profiles of mature leaves revealed that several biosynthetic pathways downstream of pyruvate are altered in re and rer3. Double mutant analysis showed that only re rer1 and rer5 rer6 exhibited synergistic phenotypes, indicating functional redundancy. The redundancy between RE and its closest paralog, RER1, was confirmed by overexpressing RER1 in re mutants, which partially suppressed leaf reticulation. Our results show that RER family members can be divided into four functional modules with divergent functions. Moreover, these results provide insights into the origin of the reticulated phenotype, suggesting that the RER proteins functionally interconnect photoperiodic growth, amino acid homeostasis, and reactive oxygen species metabolism during Arabidopsis leaf growth.

Understanding metabolic acclimation of plants to challenging environmental conditions is essential for dissecting the role of metabolic pathways in growth and survival. As stresses involve simultaneous physiological alterations across all levels of cellular organization, a comprehensive characterization of the role of metabolic pathways in acclimation necessitates integration of genome-scale models with high-throughput data. Here, we present an integrative optimization-based approach, which, by coupling a plant metabolic network model and transcriptomics data, can predict the metabolic pathways affected in a single, carefully controlled experiment. Moreover, we propose three optimization-based indices that characterize different aspects of metabolic pathway behavior in the context of the entire metabolic network. We demonstrate that the proposed approach and indices facilitate quantitative comparisons and characterization of the plant metabolic response under eight different light and/or temperature conditions. The predictions of the metabolic functions involved in metabolic acclimation of Arabidopsis thaliana to the changing conditions are in line with experimental evidence and result in a hypothesis about the role of homocysteine-to-Cys interconversion and Asn biosynthesis. The approach can also be used to reveal the role of particular metabolic pathways in other scenarios, while taking into consideration the entirety of characterized plant metabolism.

Regulation of metabolism at the level of transcription and its corollary metabolite-mediated regulation of transcription are well-documented mechanisms by which plants adapt to circumstance. That said the function of only a minority of transcription factor networks are fully understood and it seems likely that we have only identified a subset of the metabolites that play a mediator function in the regulation of transcription. Here we describe an integrated genomics approach in which we perform combined transcript and metabolite profiling on Arabidopsis (Arabidopsis thaliana) plants challenged by various environmental extremes. We chose this approach to generate a large variance in the levels of all parameters recorded. The data was then statistically evaluated to identify metabolites whose level robustly correlated with those of a particularly large number of transcripts. Since correlation alone provides no proof of causality we subsequently attempted to validate these putative mediators of gene expression via a combination of statistical analysis of data available in publicly available databases and iterative experimental evaluation. Data presented here suggest that, on adoption of appropriate caution, the approach can be used for the identification of metabolite mediators of gene expression. As an exemplary case study we document that in plants, as in yeast (Saccharomyces cerevisiae) and mammals, leucine plays an important role as a regulator of gene expression and provide a leucine response gene regulatory network.