Sergey Malitsky

Reconstruction of Arabidopsis metabolic network models accounting for subcellular compartmentalization and tissue-specificity.

Plant metabolic engineering is commonly used in the production of functional foods and quality trait improvement. However, to date, computational model-based approaches have only been scarcely used in this important endeavor, in marked contrast to their prominent success in microbial metabolic engineering. In this study we present a computational pipeline for the reconstruction of fully compartmentalized tissue-specific models of Arabidopsis thaliana on a genome scale. This reconstruction involves automatic extraction of known biochemical reactions in Arabidopsis for both primary and secondary metabolism, automatic gap-filling, and the implementation of methods for determining subcellular localization and tissue assignment of enzymes. The reconstructed tissue models are amenable for constraint-based modeling analysis, and significantly extend upon previous model reconstructions. A set of computational validations (i.e., cross-validation tests, simulations of known metabolic functionalities) and experimental validations (comparison with experimental metabolomics datasets under various compartments and tissues) strongly testify to the predictive ability of the models. The utility of the derived models was demonstrated in the prediction of measured fluxes in metabolically engineered seed strains and the design of genetic manipulations that are expected to increase vitamin E content, a significant nutrient for human health. Overall, the reconstructed tissue models are expected to lay down the foundations for computational-based rational design of plant metabolic engineering. The reconstructed compartmentalized Arabidopsis tissue models are MIRIAM-compliant and are available upon request.

Biosynthesis of Antinutritional Alkaloids in Solanaceous Crops Is Mediated by Clustered Genes

From Nasty to Tasty Some of our favorite food crops derive from wild relatives that were distasteful or even toxic. Domestication over many years selected for variants with reduced levels of antinutritional compounds. The wild relatives remain valuable, however, for other traits such as resistance to pathogens, but their use in crop development is complicated by the continued presence of unpalatable compounds. Itkin et al. (p. 175, published online 20 June) elucidate the metabolic pathways and genes directing synthesis of some of these antinutritionals in potato and tomato. Some of the chemicals that domestication has reduced in potato and tomato are derived from clusters of biosynthetic genes. Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants—as, for example, potato—are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.

The Transcript and Metabolite Networks Affected by the Two Clades of Arabidopsis Glucosinolate Biosynthesis Regulators

In this study, transcriptomics and metabolomics data were integrated in order to examine the regulation of glucosinolate (GS) biosynthesis in Arabidopsis (Arabidopsis thaliana) and its interface with pathways of primary metabolism. Our genetic material for analyses were transgenic plants overexpressing members of two clades of genes (ALTERED TRYPTOPHAN REGULATION1 [ATR1]-like and MYB28-like) that regulate the aliphatic and indole GS biosynthetic pathways (AGs and IGs, respectively). We show that activity of these regulators is not restricted to the metabolic space surrounding GS biosynthesis but is tightly linked to more distal metabolic networks of primary metabolism. This suggests that with similarity to the regulators we have investigated here, other factors controlling pathways of secondary metabolism might also control core pathways of central metabolism. The relatively broad view of transcripts and metabolites altered in transgenic plants overexpressing the different factors underlined novel links of GS metabolism to additional metabolic pathways, including those of jasmonic acid, folate, benzoic acid, and various phenylpropanoids. It also revealed transcriptional and metabolic hubs in the “distal” network of metabolic pathways supplying precursors to GS biosynthesis and that overexpression of the ATR1-like clade genes has a much broader effect on the metabolism of indolic compounds than described previously. While the reciprocal, negative cross talk between the methionine and tryptophan pathways that generate GSs in Arabidopsis has been suggested previously, we now show that it is not restricted to AGs and IGs but includes additional metabolites, such as the phytoalexin camalexin. Combining the profiling data of transgenic lines with gene expression correlation analysis allowed us to propose a model of how the balance in the metabolic network is maintained by the GS biosynthesis regulators. It appears that ATR1/MYB34 is an important mediator between the gene activities of the two clades. While it is very similar to the ATR1-like clade members in terms of downstream gene targets, its expression is highly correlated with that of the MYB28-like clade members. Finally, we used the unique transgenic plants obtained here to show that AGs are likely more potent deterrents of the whitefly Bemisia tabaci compared with IGs. The influence on insect behavior raises an important question for future investigation of the functional aspect of our initial finding, which pointed to enriched expression of the MYB28-like clade genes in the abaxial domain of the Arabidopsis leaf.

Persistent microbiome alterations modulate the rate of post-dieting weight regain

In tackling the obesity pandemic, considerable efforts are devoted to the development of effective weight reduction strategies, yet many dieting individuals fail to maintain a long-term weight reduction, and instead undergo excessive weight regain cycles. The mechanisms driving recurrent post-dieting obesity remain largely elusive. Here we identify an intestinal microbiome signature that persists after successful dieting of obese mice and contributes to faster weight regain and metabolic aberrations upon re-exposure to obesity-promoting conditions. Faecal transfer experiments show that the accelerated weight regain phenotype can be transmitted to germ-free mice. We develop a machine-learning algorithm that enables personalized microbiome-based prediction of the extent of post-dieting weight regain. Additionally, we find that the microbiome contributes to diminished post-dieting flavonoid levels and reduced energy expenditure, and demonstrate that flavonoid-based ‘post-biotic’ intervention ameliorates excessive secondary weight gain. Together, our data highlight a possible microbiome contribution to accelerated post-dieting weight regain, and suggest that microbiome-targeting approaches may help to diagnose and treat this common disorder.

GLYCOALKALOID METABOLISM1 Is Required for Steroidal Alkaloid Glycosylation and Prevention of Phytotoxicity in Tomato

Steroidal alkaloids (SAs) are specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. In this study, the role of GLYCOALKALOID METABOLISM1 (GAME1) in the biosynthesis of tomato SAs was revealed, highlighting the importance of GAME1 in SA glycosylation and in reducing the toxicity of SA metabolites to the plant cell. Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.

High-resolution metabolic mapping of cell types in plant roots

Significance Analyzing metabolite composition offers a powerful tool for understanding gene function and regulatory processes. Here, we present nontargeted metabolomics assays of five Arabidopsis GFP-tagged lines representing core cell types in the plant root, providing a metabolic map of an organ, composed of its different cell types. Fifty metabolites were putatively identified. The most prominent groups were glucosinolates, phenylpropanoids, and dipeptides. Metabolites were differentially abundant across root cell types and in many cases, this abundance did not correlate with transcript expression, suggesting non–cell-autonomous mechanisms responsible for their targeted localization. Metabolite composition offers a powerful tool for understanding gene function and regulatory processes. However, metabolomics studies on multicellular organisms have thus far been performed primarily on whole organisms, organs, or cell lines, losing information about individual cell types within a tissue. With the goal of profiling metabolite content in different cell populations within an organ, we used FACS to dissect GFP-marked cells from Arabidopsis roots for metabolomics analysis. Here, we present the metabolic profiles obtained from five GFP-tagged lines representing core cell types in the root. Fifty metabolites were putatively identified, with the most prominent groups being glucosinolates, phenylpropanoids, and dipeptides, the latter of which is not yet explored in roots. The mRNA expression of enzymes or regulators in the corresponding biosynthetic pathways was compared with the relative metabolite abundance. Positive correlations suggest that the rate-limiting steps in biosynthesis of glucosinolates in the root are oxidative modifications of side chains. The current study presents a work flow for metabolomics analyses of cell-type populations.

Sugar Synthesis from CO2 in Escherichia coli

Summary Can a heterotrophic organism be evolved to synthesize biomass from CO2 directly? So far, non-native carbon fixation in which biomass precursors are synthesized solely from CO2 has remained an elusive grand challenge. Here, we demonstrate how a combination of rational metabolic rewiring, recombinant expression, and laboratory evolution has led to the biosynthesis of sugars and other major biomass constituents by a fully functional Calvin-Benson-Bassham (CBB) cycle in E. coli. In the evolved bacteria, carbon fixation is performed via a non-native CBB cycle, while reducing power and energy are obtained by oxidizing a supplied organic compound (e.g., pyruvate). Genome sequencing reveals that mutations in flux branchpoints, connecting the non-native CBB cycle to biosynthetic pathways, are essential for this phenotype. The successful evolution of a non-native carbon fixation pathway, though not yet resulting in net carbon gain, strikingly demonstrates the capacity for rapid trophic-mode evolution of metabolism applicable to biotechnology. PaperClip

Mapping the diatom redox-sensitive proteome provides insight into response to nitrogen stress in the marine environment

Significance Phytoplankton form massive blooms in the oceans that are controlled by nutrients, light availability, and biotic interactions with grazers and viruses. Although phytoplankton were traditionally considered passive drifters with currents here we demonstrate how diatom cells sense and respond to oxidative stress through a redox-sensitive protein network. We further demonstrate the redox sensitivity of nitrogen assimilation, which is essential for diatom blooms in the ocean, and provide compelling evidence for organelle-specific oxidation patterns under nitrogen stress conditions using a genetically encoded redox sensor. We propose that redox regulation of metabolic rates in the response to stress provides a mechanism of acclimation to rapid fluctuations in the chemophysical gradients in the marine environment. Diatoms are ubiquitous marine photosynthetic eukaryotes responsible for approximately 20% of global photosynthesis. Little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a quantitative mass spectrometry-based approach to elucidate the redox-sensitive signaling network (redoxome) mediating the response of diatoms to oxidative stress. We quantified the degree of oxidation of 3,845 cysteines in the Phaeodactylum tricornutum proteome and identified approximately 300 redox-sensitive proteins. Intriguingly, we found redox-sensitive thiols in numerous enzymes composing the nitrogen assimilation pathway and the recently discovered diatom urea cycle. In agreement with this finding, the flux from nitrate into glutamine and glutamate, measured by the incorporation of 15N, was strongly inhibited under oxidative stress conditions. Furthermore, by targeting the redox-sensitive GFP sensor to various subcellular localizations, we mapped organelle-specific oxidation patterns in response to variations in nitrogen quota and quality. We propose that redox regulation of nitrogen metabolism allows rapid metabolic plasticity to ensure cellular homeostasis, and thus is essential for the ecological success of diatoms in the marine ecosystem.

The tomato SlSHINE3 transcription factor regulates fruit cuticle formation and epidermal patterning

Fleshy tomato fruit typically lacks stomata; therefore, a proper cuticle is particularly vital for fruit development and interaction with the surroundings. Here, we characterized the tomato SlSHINE3 (SlSHN3) transcription factor to extend our limited knowledge regarding the regulation of cuticle formation in fleshy fruits. We created SlSHN3 overexpressing and silenced plants, and used them for detailed analysis of cuticular lipid compositions, phenotypic characterization, and the study on the mode of SlSHN3 action. Heterologous expression of SlSHN3 in Arabidopsis phenocopied overexpression of the Arabidopsis SHNs. Silencing of SlSHN3 results in profound morphological alterations of the fruit epidermis and significant reduction in cuticular lipids. We demonstrated that SlSHN3 activity is mediated by control of genes associated with cutin metabolism and epidermal cell patterning. As with SlSHN3 RNAi lines, mutation in the SlSHN3 target gene, SlCYP86A69, resulted in severe cutin deficiency and altered fruit surface architecture. In vitro activity assays demonstrated that SlCYP86A69 possesses NADPH-dependent ω-hydroxylation activity, particularly of C18:1 fatty acid to the 18-hydroxyoleic acid cutin monomer. This study provided insights into transcriptional mechanisms mediating fleshy fruit cuticle formation and highlighted the link between cutin metabolism and the process of fruit epidermal cell patterning.

Expression of a bacterial bi‐functional chorismate mutase/prephenate dehydratase modulates primary and secondary metabolism associated with aromatic amino acids in Arabidopsis

Plants can synthesize the aromatic amino acid Phe via arogenate, but it is still not known whether they also use an alternative route for Phe biosynthesis via phenylpyruvate, like many micro-organisms. To examine this possibility, we expressed a bacterial bi-functional PheA (chorismate mutase/prephenate dehydratase) gene in Arabidopsis thaliana that converts chorismate via prephenate into phenylpyruvate. The PheA-expressing plants showed a large increase in the level of Phe, implying that they can convert phenylpyruvate into Phe. In addition, PheA expression rendered the plants more sensitive than wild-type plants to the Trp biosynthesis inhibitor 5-methyl-Trp, implying that Phe biosynthesis competes with Trp biosynthesis from their common precursor chorismate. Surprisingly, GC-MS, LC-MS and microarray analyses showed that this increase in Phe accumulation only had a very minor effect on the levels of other primary metabolites as well as on the transcriptome profile, implying little regulatory cross-interaction between the aromatic amino acid biosynthesis network and the bulk of the Arabidopsis transcriptome and primary metabolism. However, the levels of a number of secondary metabolites derived from all three aromatic amino acids (Phe, Trp and Tyr) were altered in the PheA plants, implying regulatory cross-interactions between the flux of aromatic amino acid biosynthesis from chorismate and their further metabolism into various secondary metabolites. Taken together, our results provide insights into the regulatory mechanisms of aromatic amino acid biosynthesis and their interaction with central primary metabolism, as well as the regulatory interface between primary and secondary metabolism.