Ontologies for increasing the FAIRness of plant research data.

The importance of improving the FAIRness (findability, accessibility, interoperability, reusability) of research data is undeniable, especially in the face of large, complex datasets currently being produced by omics technologies. Facilitating the integration of a dataset with other types of data increases the likelihood of reuse, and the potential of answering novel research questions. Ontologies are a useful tool for semantically tagging datasets as adding relevant metadata increases the understanding of how data was produced and increases its interoperability. Ontologies provide concepts for a particular domain as well as the relationships between concepts. By tagging data with ontology terms, data becomes both human- and machine- interpretable, allowing for increased reuse and interoperability. However, the task of identifying ontologies relevant to a particular research domain or technology is challenging, especially within the diverse realm of fundamental plant research. In this review, we outline the ontologies most relevant to the fundamental plant sciences and how they can be used to annotate data related to plant-specific experiments within metadata frameworks, such as Investigation-Study-Assay (ISA). We also outline repositories and platforms most useful for identifying applicable ontologies or finding ontology terms.

Mapping the castor bean endosperm proteome revealed a metabolic interaction between plastid, mitochondria, and peroxisomes to optimize seedling growth.

In this work, we studied castor-oil plant Ricinus communis as a classical system for endosperm reserve breakdown. The seeds of castor beans consist of a centrally located embryo with the two thin cotyledons surrounded by the endosperm. The endosperm functions as major storage tissue and is packed with nutritional reserves, such as oil, proteins, and starch. Upon germination, mobilization of the storage reserves requires inter-organellar interplay of plastids, mitochondria, and peroxisomes to optimize growth for the developing seedling. To understand their metabolic interactions, we performed a large-scale organellar proteomic study on castor bean endosperm. Organelles from endosperm of etiolated seedlings were isolated and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Computer-assisted deconvolution algorithms were applied to reliably assign the identified proteins to their correct subcellular localization and to determine the abundance of the different organelles in the heterogeneous protein samples. The data obtained were used to build a comprehensive metabolic model for plastids, mitochondria, and peroxisomes during storage reserve mobilization in castor bean endosperm.

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.

Carbon monoxide exposure activates ULK1 via AMPK phosphorylation in murine embryonic fibroblasts.

Carbon monoxide (CO) is endogenously produced upon degradation of heme by heme oxygenases (HOs) and is suggested to act as a gaseous signaling molecule. The expression of HO-1 is triggered by the Nrf2-Keap1 signaling pathway which responds to exogenous stress signals and dietary constituents such as flavonoids and glucosinolates or reactive metabolic intermediates like 4-hydroxynonenal. Endogenous CO affects energy metabolism, regulates the utilization of glucose and addresses CYP450 enzymes. Using the CO releasing molecule-401 (CORM-401), we studied the effect of endogenous CO on ATP synthesis, AMP-signaling and activation of the AMPK pathway in cell culture. Upon exposure of cells to CORM-401, the mitochondrial ATP production rate was significantly decreased (P=0.007) to about 50%, while glycolytic ATP synthesis was unchanged (P=0.489). Total ATP levels were less affected as determined by mass spectrometry. Instead, levels of ADP and AMP were elevated following CORM-401 exposure by about two- (P=0.022) and four-fold (P=0.012) compared to control, respectively. Increased concentrations of AMP activate AMPK which was demonstrated by a 10 to 15-fold increased phosphorylation of Thr172 of the α-subunit of AMPK (P=0.025). A downstream target of AMPK is the kinase ULK1 which triggers autophagic and mitophagic processes. Activation of ULK1 after CO exposure was proven by a 3 to 5-fold elevated phosphorylation of ULK1 at Ser555 (P=0.004). The present data suggest that production of endogenous CO leads to increasing amounts of AMP which mediates AMPK-dependent downstream effects and likely triggers autophagic processes. Since dietary constituents and their metabolites induce the expression of the CO producing enzyme HO-1, CO signaling may also be involved in the cellular response to nutritional factors.

Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner.

Astrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.

Endogenous Carbon Monoxide Signaling Modulates Mitochondrial Function and Intracellular Glucose Utilization: Impact of the Heme Oxygenase Substrate Hemin.

Stress-inducible heme oxygenase-1 (HO-1) catalyzes the oxidative cleavage of heme yielding biliverdin, ferrous iron, and carbon monoxide (CO). Heme oxygenase activity has been attributed to antioxidant defense via the redox cycling system of biliverdin and bilirubin. There is increasing evidence that CO is a gaseous signaling molecule and plays a role in the regulation of energy metabolism. Inhibitory effects of CO on the respiratory chain are well established, but the implication of such a process on the cellular stress response is not well understood. By means of extracellular flux analyses and isotopic tracing, we studied the effects of CO, either released from the CO donor CORM-401 or endogenously produced by heme oxygenases, on the respiratory chain and glucose metabolism. CORM-401 was thereby used as a tool to mimic endogenous CO production by heme oxygenases. In the long term (>60 min), CORM-401-derived CO exposure inhibited mitochondrial respiration, which was compensated by increased glycolysis accompanied by a loss of the ATP production rate and an increase in proton leakage. This effect pattern was likewise observed after endogenous CO production by heme oxygenases. However, in the present setting, these effects were only observed when sufficient substrate for heme oxygenases (hemin) was provided. Modulation of the HO-1 protein level was less important. The long-term influence of CO on glucose metabolism via glycolysis was preceded by a short-term response (<30 min) of the cells to CO. Stable isotope-labeling experiments and metabolic flux analysis revealed a short-term shift of glucose consumption from glycolysis to the pentose phosphate pathway (PPP) along with an increase in reactive oxygen species (ROS) generation. Overall, we suggest that signaling by endogenous CO stimulates the rapid formation of reduction equivalents (NADPH) via the PPP, and plays an additional role in antioxidant defense, e.g., via feed-forward stimulation of the bilirubin/biliverdin redox cycling system.

Molecular plant responses to combined abiotic stresses put a spotlight on unknown and abundant genes.

Environmental stresses such as drought, heat, and salinity limit plant development and agricultural productivity. While individual stresses have been studied extensively, much less is known about the molecular interaction of responses to multiple stresses. To address this problem, we investigated molecular responses of Arabidopsis to single, double, and triple combinations of salt, osmotic, and heat stresses. A metabolite profiling analysis indicated the production of specific compatible solutes depending on the nature of the stress applied. We found that in combination with other stresses, heat has a dominant effect on global gene expression and metabolite level patterns. Treatments that include heat stress lead to strongly reduced transcription of genes coding for abundant photosynthetic proteins and proteins regulating the cell life cycle, while genes involved in protein degradation are up-regulated. Under combined stress conditions, the plants shifted their metabolism to a survival state characterized by low productivity. Our work provides molecular evidence for the dangers for plant productivity and future world food security posed by heat waves resulting from global warming. We highlight candidate genes, many of which are functionally uncharacterized, for engineering plant abiotic stress tolerance.

Biochemical and functional characterization of a mitochondrial citrate carrier in Arabidopsis thaliana.

A homolog of the mitochondrial succinate/fumarate carrier from yeast (Sfc1p) has been found in the Arabidopsis genome, named AtSFC1. The AtSFC1 gene was expressed in Escherichia coli, and the gene product was purified and reconstituted in liposomes. Its transport properties and kinetic parameters demonstrated that AtSFC1 transports citrate, isocitrate and aconitate and, to a lesser extent, succinate and fumarate. This carrier catalyzes a fast counter-exchange transport as well as a low uniport of substrates, exhibits a higher transport affinity for tricarboxylates than dicarboxylates, and is inhibited by pyridoxal 5'-phosphate and other inhibitors of mitochondrial carriers to various degrees. Gene expression analysis indicated that the AtSFC1 transcript is mainly present in heterotrophic tissues, and fusion with a green-fluorescent protein localized AtSFC1 to the mitochondria. Furthermore, 35S-AtSFC1 antisense lines were generated and characterized at metabolic and physiological levels in different organs and at various developmental stages. Lower expression of AtSFC1 reduced seed germination and impaired radicle growth, a phenotype that was related to reduced respiration rate. These findings demonstrate that AtSFC1 might be involved in storage oil mobilization at the early stages of seedling growth and in nitrogen assimilation in root tissue by catalyzing citrate/isocitrate or citrate/succinate exchanges.

Aroma and quality of breads baked from old and modern wheat varieties and their prediction from genomic and flour-based metabolite profiles.

Bread aroma is the principal characteristic perceived by the consumer yet it is mostly disregarded in the product chain. The main aim of this study was to evaluate the potential to include bread aroma as a new target criterion into the wheat product chain. The objectives of our study were to (i) quantify the influence of genetic versus environmental factors on the bread aroma and quality characteristics, (ii) evaluate whether bread baked from modern wheat varieties differ in terms of aroma from those baked from old varieties, and (iii) compare genomic and metabolomic approaches for their efficiency to predict bread aroma and quality characteristics in a wheat breeding program. Agronomic characters as well as bread aroma and quality traits were assessed for 18 old and 22 modern winter wheat varieties evaluated at up to three locations in Germany. Metabolite profiles of all 120 flour samples were collected using a 7200 GC-QTOF. Considerable differences in the adjusted entry means for all examined bread aroma and quality characters were observed. For aroma, which was rated on a scale from 1 to 9, the adjusted entry means varied for the 40 wheat varieties between 3 and 8. In contrast, the aroma of bread prepared from old and modern wheat varieties did not differ significantly (P < 0.05). Bread aroma was not significantly (P < 0.05) correlated with grain yield, which suggested that it is possible to select for the former character in wheat breeding programs without reducing the gain of selection for the latter. Finally, we have shown that bread aroma can be better predicted using a combination of metabolite and SNP genotyping profiles instead of the SNP genotyping profile only. In conclusion, we have illustrated possibilities to increase the quality of wheat for consumers in the product chain.

Transcript and metabolite changes during the early phase of abscisic acid-mediated induction of crassulacean acid metabolism in Talinum triangulare.

Crassulacean acid metabolism (CAM) has evolved as a water-saving strategy, and its engineering into crops offers an opportunity to improve their water use efficiency. This requires a comprehensive understanding of the regulation of the CAM pathway. Here, we use the facultative CAM species Talinum triangulare as a model in which CAM can be induced rapidly by exogenous abscisic acid. RNA sequencing and metabolite measurements were employed to analyse the changes underlying CAM induction and identify potential CAM regulators. Non-negative matrix factorization followed by k-means clustering identified an early CAM-specific cluster and a late one, which was specific for the early light phase. Enrichment analysis revealed abscisic acid metabolism, WRKY-regulated transcription, sugar and nutrient transport, and protein degradation in these clusters. Activation of the CAM pathway was supported by up-regulation of phosphoenolpyruvate carboxylase, cytosolic and chloroplastic malic enzymes, and several transport proteins, as well as by increased end-of-night titratable acidity and malate accumulation. The transcription factors HSFA2, NF-YA9, and JMJ27 were identified as candidate regulators of CAM induction. With this study we promote the model species T. triangulare, in which CAM can be induced in a controlled way, enabling further deciphering of CAM regulation.

Corrigendum: Knock-Down of the Phosphoserine Phosphatase Gene Effects Rather N- Than S-Metabolism in Arabidopsis thaliana.

[This corrects the article DOI: 10.3389/fpls.2018.01830.].

Loss of Function of Rice Plastidic Glycolate/Glycerate Translocator 1 Impairs Photorespiration and Plant Growth.

Ribulose-1,5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic carbon fixation, is able to accept both O2 and CO2 as substrates. When it fixes O2, it produces 2-phosphoglycolate, which is detoxified by photorespiration and recycled to the Calvin-Benson-Bassham cycle. To complete photorespiration, metabolite transport across three organelles, chloroplasts, peroxisomes, and mitochondria, is necessary through transmembrane transporters. In rice (Oryza sativa) little is known about photorespiratory transmembrane transporters. Here, we identified the rice plastidic glycolate/glycerate translocator 1 (OsPLGG1), a homolog of Arabidopsis PLGG1. OsPLGG1 mutant lines, osplgg1-1, osplgg1-2, and osplgg1-3, showed a growth retardation phenotype, such as pale green leaf, reduced tiller number, and reduced seed grain weight as well as reduced photosynthetic carbon reduction rate due to low activities of photosystem I and II. The plant growth retardation in osplgg1 mutants was rescued under high CO2 condition. Subcellular localization of OsPLGG1-GFP fusion protein, along with its predicted N-terminal transmembrane domain, confirmed that OsPLGG1 is a chloroplast transmembrane protein. Metabolite analysis indicated significant accumulation of photorespiratory metabolites, especially glycolate and glycerate, which have been shown to be transported by the Arabidopsis PLGG1, and changes for a number of metabolites which are not intermediates of photorespiration in the mutants. These results suggest that OsPLGG1 is the functional plastidic glycolate/glycerate transporter, which is necessary for photorespiration and growth in rice.

The Photorespiratory BOU Gene Mutation Alters Sulfur Assimilation and Its Crosstalk With Carbon and Nitrogen Metabolism in Arabidopsis thaliana.

This study was aimed at elucidating the significance of photorespiratory serine (Ser) production for cysteine (Cys) biosynthesis. For this purpose, sulfur (S) metabolism and its crosstalk with nitrogen (N) and carbon (C) metabolism were analyzed in wildtype Arabidopsis and its photorespiratory bou-2 mutant with impaired glycine decarboxylase (GDC) activity. Foliar glycine and Ser contents were enhanced in the mutant at day and night. The high Ser levels in the mutant cannot be explained by transcript abundances of genes of the photorespiratory pathway or two alternative pathways of Ser biosynthesis. Despite enhanced foliar Ser, reduced GDC activity mediated a decline in sulfur flux into major sulfur pools in the mutant, as a result of deregulation of genes of sulfur reduction and assimilation. Still, foliar Cys and glutathione contents in the mutant were enhanced. The use of Cys for methionine and glucosinolates synthesis was reduced in the mutant. Reduced GDC activity in the mutant downregulated Calvin Cycle and nitrogen assimilation genes, upregulated key enzymes of glycolysis and the tricarboxylic acid (TCA) pathway and modified accumulation of sugars and TCA intermediates. Thus, photorespiratory Ser production can be replaced by other metabolic Ser sources, but this replacement deregulates the cross-talk between S, N, and C metabolism.

Knock-Down of the Phosphoserine Phosphatase Gene Effects Rather N- Than S-Metabolism in Arabidopsis thaliana.

The aim of present study was to elucidate the significance of the phosphorylated pathway of Ser production for Cys biosynthesis in leaves at day and night and upon cadmium (Cd) exposure. For this purpose, Arabidopsis wildtype plants as control and its psp mutant knocked-down in phosphoserine phosphatase (PSP) were used to test if (i) photorespiratory Ser is the dominant precursor of Cys synthesis in autotrophic tissue in the light, (ii) the phosphorylated pathway of Ser production can take over Ser biosynthesis in leaves at night, and (iii) Cd exposure stimulates Cys and glutathione (GSH) biosynthesis and effects the crosstalk of S and N metabolism, irrespective of the Ser source. Glycine (Gly) and Ser contents were not affected by reduction of the psp transcript level confirming that the photorespiratory pathway is the main route of Ser synthesis. The reduction of the PSP transcript level in the mutant did not affect day/night regulation of sulfur fluxes while day/night fluctuation of sulfur metabolite amounts were no longer observed, presumably due to slower turnover of sulfur metabolites in the mutant. Enhanced contents of non-protein thiols in both genotypes and of GSH only in the psp mutant were observed upon Cd treatment. Mutation of the phosphorylated pathway of Ser biosynthesis caused an accumulation of alanine, aspartate, lysine and a decrease of branched-chain amino acids. Knock-down of the PSP gene induced additional defense mechanisms against Cd toxicity that differ from those of WT plants.

Reversible Burst of Transcriptional Changes during Induction of Crassulacean Acid Metabolism in Talinum triangulare.

Drought tolerance is a key factor for agriculture in the 21st century as it is a major determinant of plant survival in natural ecosystems as well as crop productivity. Plants have evolved a range of mechanisms to cope with drought, including a specialized type of photosynthesis termed Crassulacean acid metabolism (CAM). CAM is associated with stomatal closure during the day as atmospheric CO2 is assimilated primarily during the night, thus reducing transpirational water loss. The tropical herbaceous perennial species Talinum triangulare is capable of transitioning, in a facultative, reversible manner, from C3 photosynthesis to weakly expressed CAM in response to drought stress. The transcriptional regulation of this transition has been studied. Combining mRNA-Seq with targeted metabolite measurements, we found highly elevated levels of CAM-cycle enzyme transcripts and their metabolic products in T. triangulare leaves upon water deprivation. The carbohydrate metabolism is rewired to reduce the use of reserves for growth to support the CAM-cycle and the synthesis of compatible solutes. This large-scale expression dataset of drought-induced CAM demonstrates transcriptional regulation of the C3-CAM transition. We identified candidate transcription factors to mediate this photosynthetic plasticity, which may contribute in the future to the design of more drought-tolerant crops via engineered CAM.