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Summary: Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched‐chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant‐derived foods contain only small amounts of BCAAs, and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide‐resistant version of an acetolactate synthase (mSlALS) gene expressed broadly and a MYB12‐like transcription factor (SlMYB12) expressed in a fruit‐specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21‐fold compared to wild‐type levels), Valine (ninefold) and Isoleucine (threefold) and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64‐fold) and quercetin (45‐fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption. [ABSTRACT FROM AUTHOR]

SUMMARY: Programmable transcriptional regulators based on CRISPR architecture are promising tools for the induction of plant gene expression. In plants, CRISPR gene activation is effective with respect to modulating development processes, such as the flowering time or customizing biochemical composition. The most widely used method for delivering CRISPR components into the plant is Agrobacterium tumefaciens‐mediated genetic transformation, either transient or stable. However, as a result of their versatility and their ability to move, virus‐derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular guide RNA (gRNA), which represents the variable component in CRISPR strategies. In the present study, we describe a Potato virus X‐derived vector that, upon agroinfection in Nicotiana benthamiana, serves as a vehicle for delivery of gRNAs, producing highly specific virus‐induced gene activation. The system works in combination with a N. benthamiana transgenic line carrying the remaining complementary CRISPR gene activation components, specifically the dCasEV2.1 cassette, which has been shown previously to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non‐invasive spraying method, we show that gRNA‐mediated activation programs move locally and systemically, generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this Potato virus X‐based virus‐induced gene reprogramming strategy results in program‐specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid‐enriched metabolite profiles. Significance Statement: The virus‐derived systems for delivering CRISPR components to the plants have emerged as alternatives to the classical methods of plant transformation as a result of their versatility and their movement capacity. The presented PVX_VIGR approach provides an optimized CRISPR gene activation‐guide RNA delivery that expands the toolbox for reprogramming gene expression in plants. In addition, an easily scalable, non‐invasive spraying optimized method for PVX_VIGR strategy results in a specific modulation of the metabolic content in N. benthamiana. [ABSTRACT FROM AUTHOR]

SUMMARY: Sustainable production of chemicals and improving these biosources by engineering metabolic pathways to create efficient plant‐based biofactories relies on the knowledge of available chemical/biosynthetic diversity present in the plant. Nicotiana species are well known for their amenability towards transformation and other new plant breeding techniques. The genus Nicotiana is primarily known through Nicotiana tabacum L., the source of tobacco leaves and all respective tobacco products. Due to the prevalence of the latter, N. tabacum and related Nicotiana species are one of the most extensively studied plants. The majority of studies focused solely on N. tabacum or other individual species for chemotyping. The present study analysed a diversity panel including 17 Nicotiana species and six accessions of Nicotiana benthamiana and created a data set that effectively represents the chemotype core collection of the genus Nicotiana. The utilisation of several analytical platforms and previously published libraries/databases enabled the identification and measurement of over 360 metabolites of a wide range of chemical classes as well as thousands of unknowns with dedicated spectral and chromatographic properties. Significance Statement: A chemotype core collection for the genus Nicotiana was compiled. The metabolite composition of 17 Nicotiana species analysed was reproducible under different growth seasons and showed a rich source of chemical diversity. The compiled database catalogued over 360 identified metabolites and thousands of unknown chemical features. These data highlight Nicotiana species with enhanced levels of high‐value chemicals and of precursors utilised for pathway engineering of high‐value chemicals in a renewable manner. [ABSTRACT FROM AUTHOR]

Summary: Roots, tubers, and bananas (RTB) are vital staples for food security in the world's poorest nations. A major constraint to current RTB breeding programmes is limited knowledge on the available diversity due to lack of efficient germplasm characterization and structure. In recent years large‐scale efforts have begun to elucidate the genetic and phenotypic diversity of germplasm collections and populations and, yet, biochemical measurements have often been overlooked despite metabolite composition being directly associated with agronomic and consumer traits. Here we present a compound database and concentration range for metabolites detected in the major RTB crops: banana (Musa spp.), cassava (Manihot esculenta), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), and yam (Dioscorea spp.), following metabolomics‐based diversity screening of global collections held within the CGIAR institutes. The dataset including 711 chemical features provides a valuable resource regarding the comparative biochemical composition of each RTB crop and highlights the potential diversity available for incorporation into crop improvement programmes. Particularly, the tropical crops cassava, sweet potato and banana displayed more complex compositional metabolite profiles with representations of up to 22 chemical classes (unknowns excluded) than that of potato, for which only metabolites from 10 chemical classes were detected. Additionally, over 20% of biochemical signatures remained unidentified for every crop analyzed. Integration of metabolomics with the on‐going genomic and phenotypic studies will enhance 'omics‐wide associations of molecular signatures with agronomic and consumer traits via easily quantifiable biochemical markers to aid gene discovery and functional characterization. Significance Statement: A metabolite‐specific database cataloguing the biochemical diversity within and between root, tuber, and banana (RTB) crops has been compiled from profiling thousands of accessions. The database records the extent of metabolite concentrations available in screened germplasm of each RTB crop and therefore can be used to set breeding targets. This information aids in crop breeding programmes to improve the livelihoods for more than two billion people reliant on RTB crops. [ABSTRACT FROM AUTHOR]

Dwindling fossil fuel reserves and poor environmental credentials of chemical synthesis means, new renewable sources for the production and manufacture of valuable chemicals and pharmaceuticals are required. Presently, tobacco is an underutilised non-food crop with the potential to act as a biofactory. In this study, metabolite profiling across vegetative development has been carried out to provide a quantitative baseline of metabolites, their formation and interaction. Two tobacco platforms have been used, Nicotiana benthamiana and Nicotiana tabacum. Our data generated has provided the quantitative and qualitative baseline levels for exploitable pathways and metabolites, across two complementary Nicotiana species. N. benthamiana is the chassis of choice for transient expression. The metabolite data obtained for N. benthamiana highlighted that before flower emergence, the increased central carbon metabolism and high amino acid levels are available for the biosynthesis of endogenous or heterologous metabolites. In the future, engineering pathways or biocatalysts into N. benthamiana could add value to the process presently used to produce low volume, high cost pharmaceuticals. Similar outputs were obtained for N. tabacum, which has the advantage of providing a large biomass and hence, high product yield. These data provide an insight into the metabolite pools available in tobacco for future exploitation by emerging New Plant Breeding Techniques. • Qualitative and quantitative baselines for N. benthamiana and N. tabacum metabolites. • Increased central carbon metabolism and amino acids occur prior to flowering. • Nicotine levels are associated with development of reproductive organs. • Unlike N. benthamiana, total polyphenol levels fluctuate during N. tabacum development. [ABSTRACT FROM AUTHOR]

Light is an essential regulator of many developmental processes in higher plants. We investigated the effect of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1/2 genes (OsHDR1/2) and isopentenyl diphosphate isomerase 1/2 genes (OsIPPI1/2) on the biosynthesis of chlorophylls, carotenoids, and phytosterols in 14-day-old etiolated rice (Oyza sativa L.) leaves during de-etiolation. However, little is known about the effect of isoprenoid biosynthesis genes on the corresponding metabolites during the de-etiolation of etiolated rice leaves. The results showed that the levels of α-tocopherol were significantly increased in de-etiolated rice leaves. Similar to 1-deoxy-D-xylulose-5-phosphate synthase 3 gene (OsDXS3), both OsDXS1 and OsDXS2 genes encode functional 1-deoxy-D-xylulose-5-phosphate synthase (DXS) activities. Their expression patterns and the synthesis of chlorophyll, carotenoid, and tocopherol metabolites suggested that OsDXS1 is responsible for the biosynthesis of plastidial isoprenoids in de-etiolated rice leaves. The expression analysis of isoprenoid biosynthesis genes revealed that the coordinated expression of the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, chlorophyll, carotenoid, and tocopherol pathway genes mirrored the changes in the levels of the corresponding metabolites during de-etiolation. The underpinning mechanistic basis of coordinated light-upregulated gene expression was elucidated during the de-etiolation process, specifically the role of light-responsive cis-regulatory motifs in the promoter region of these genes. In silico promoter analysis showed that the light-responsive cis-regulatory elements presented in all the promoter regions of each light-upregulated gene, providing an important link between observed phenotype during de-etiolation and the molecular machinery controlling expression of these genes. [ABSTRACT FROM AUTHOR]

Over the previous decades, biotechnological innovations have led to improved agricultural productivity, more nutritious foods and lower chemical usage. Both in western societies and Low Medium Income Countries (LMICs). However, the projected increases in the global population, means the production of nutritious food stuffs must increase dramatically. Building on existing genetic modification technologies a series of New Plant Breeding Technologies (NPBT) has recently emerged. These approaches include, Agro-infiltration, grafting, cis and intragenesis and gene editing technologies. How these new techniques should be regulated has fostered considerable debate. Concerns have also been raised, to ensure over-regulation does not arise, creating administrative and economic burden. In this article the existing landscape of genetically modified crops is reviewed and the potential of several New Plant Breeding Techniques (NPBT) described. Metabolomics is an omic technology that has developed in a concurrent manner with biotechnological advances in plant breeding. There is potentially further opportunities to advance our metabolomic technologies to characterise the outputs of New Plant Breeding Technologies, in a manner that is beneficial both from an academic, biosafety and industrial perspective. [ABSTRACT FROM AUTHOR]

A broad diversity of phenotypes are available within the cassava germplasm collections. The phenotypes include improved nutritional, starch or culinary root quality as well as abiotic and biotic resistance properties. Some of these traits can be found naturally occurring in cassava landraces, whereas others are the result of targeted breeding efforts. For future breeding programmes it is important to know the underlying mechanisms of these desirable traits. Metabolomics can assist in the elucidation of these mechanisms by measuring the end products of the cellular processes conferring the traits of interest. The present study focused on the comparison of two or more variants of the same trait such as high and low culinary quality or resistance and susceptibility to thrips. Overall, eight different traits were assessed. Results showed that amino acids and umami compounds were associated with superior culinary attributes and the phenylpropanoid superpathway plays an important role in pest resistance. Furthermore, the data highlighted a low chemodiversity in African cassavas and that the source-sink relation was still active at the harvest stage. [ABSTRACT FROM AUTHOR]

Over the last decades, the prevalence of drug-resistance in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has increased. These findings have rekindled interest in elucidating the unique adaptive molecular and biochemistry physiology of Mycobacterium. The use of metabolite profiling independently or in combination with other levels of "-omic" analyses has proven an effective approach to elucidate key physiological/biochemical mechanisms associated with Mtb throughout infection. The following review discusses the use of metabolite profiling in the study of tuberculosis, future approaches, and the technical and logistical limitations of the methodology. [ABSTRACT FROM AUTHOR]

Mycobacterium species can cause a range of nontuberculous infections of healthy and immunocompromised people as well as infected people during and after surgical procedures. The similarity of nontuberculous mycobacteria (NTM) to the tuberculosis bacilli (TB) could ultimately enable the use of anti-TB drugs for the genus. Hence, three NTM (Mycobacterium smegmatis , Mycobacterium phlei and Mycobacterium avium) were cultured under different lab conditions, causing two mycobacterial phenotypes (active and dormant), and treated with isoniazid (INH) and ethambutol (EMB) independently or in combination. Metabolite profiling was applied to facilitate the investigation and characterisation of intracellular targets affected by the antibiotics. Aliquots of the cell culture were taken over the treatment period and the metabolite profile of the cells analysed by gas chromatography mass spectrometry. Comparative analysis of the metabolite levels to untreated mycobacteria confirmed the successful action of the antibiotics on the metabolism of all three species. Furthermore, single metabolites and metabolite pathways affected by the antibiotics could be identified and included, besides the known target sites for INH and EMB on mycobacterial cells, changes in e.g. nucleotide and saccharide levels. The combined treatment highlighted the property of EMB to enhance the effects of INH even under hypoxic culture conditions. [ABSTRACT FROM AUTHOR]

Biochemical characterisation of germplasm collections and crop wild relatives (CWRs) facilitates the assessment of biological potential and the selection of breeding lines for crop improvement. Data from the biochemical characterisation of staple root, tuber and banana (RTB) crops, i.e. banana ( Musa spp.), cassava ( Manihot esculenta ), potato ( Solanum tuberosum ), sweet potato ( Ipomoea batatas ) and yam ( Dioscorea spp .), using a metabolomics approach is presented. The data support the previously published research article "Metabolite database for root, tuber, and banana crops to facilitate modern breeding in understudied crops" (Price et al., 2020) [1]. Diversity panels for each crop, which included a variety of species, accessions, landraces and CWRs, were characterised. The biochemical profile for potato was based on five elite lines under abiotic stress. Metabolites were extracted from the tissue of foliage and storage organs (tuber, root and banana pulp) via solvent partition. Extracts were analysed via a combination of liquid chromatography - mass spectrometry (LC-MS), gas chromatography (GC)-MS, high pressure liquid chromatography with photodiode array detector (HPLC-PDA) and ultra performance liquid chromatography (UPLC)-PDA. Metabolites were identified by mass spectral matching to in-house libraries comprised from authentic standards and comparison to databases or previously published literature., Competing Interests: 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., (© 2022 Published by Elsevier Inc.)

Carotenoid biosynthesis and sequestration in higher plants occurs in the plastid organelle. Among diverse germplasm collections displaying natural variation for carotenoids and outputs from metabolic engineering experiments it has become clear that plastid type and numbers can have important implications on the quantitative composition of carotenoids accumulating. Therefore, it is important to characterize these organelles to fully evaluate the potential of the germplasm to enhance carotenoids and create nutrient dense fruits and vegetables. In this article the procedures used to isolate sub-plastidial structures from carotenoid-rich Solanaceae fruits (tomato and Capsicum) are described., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Carotenoid biosynthesis has now been subjected to metabolic engineering for over two decades. The outputs clearly show that carotenoid formation is an integral component of metabolism. Perturbations can affect intermediary metabolism and other isoprenoids. The advances in omic technologies have enabled the quantitative assessment of changes in the transcriptome, proteome and metabolome in response to altered carotenoid biosynthesis. In the present article, the approaches and procedures relating to the capture of the metabolome in response to modulation of the carotenoid biosynthetic pathway are described. These data will contribute to the fundamental understanding of metabolic biology, underpinning future rationale design of New Plant Breeding Techniques (NPBTs) and associated regulatory affairs., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cassava plays an important role as a staple food for more than 800 million people in the world due to its ability to maintain relatively high productivity even in nutrient-depleted soils. Even though cassava has been the focus of several breeding programs and has become a strong focus of research in the last few years, relatively little is currently known about its metabolism and metabolic composition in different tissues. In this article, the absolute content of sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, carotenoids, chlorophylls, tocopherols, and total protein as well as starch quality is described based on multiple analytical techniques, with protocols specifically adjusted for material from different cassava tissues. Moreover, quantification of secondary metabolites relative to internal standards is presented using both non-targeted and targeted metabolomics approaches. The protocols have also been adjusted to apply to freeze-dried material in order to allow processing of field harvest samples that typically will require long-distance transport. © 2019 The Authors. Basic Protocol 1: Metabolic profiling by gas chromatography-mass spectrometry (GC-MS) Support Protocol 1: Preparation of freeze-dried cassava material Support Protocol 2: Preparation of standard compound mixtures for absolute quantification of metabolites by GC-MS Support Protocol 3: Preparation of retention-time standard mixture Basic Protocol 2: Determination of organic acids and phosphorylated intermediates by ion chromatography-mass spectrometry (IC-MS) Support Protocol 4: Preparation of standards and recovery experimental procedure Basic Protocol 3: Determination of soluble sugars, starch, and free amino acids Alternate Protocol: Determination of soluble sugars and starch Basic Protocol 4: Determination of anions Basic Protocol 5: Determination of elements Basic Protocol 6: Determination of total protein Basic Protocol 7: Determination of non-targeted and targeted secondary metabolites Basic Protocol 8: Determination of carotenoids, chlorophylls, and tocopherol Basic Protocol 9: Determination of starch quality., (© 2019 The Authors.)

Cassava ( Manihot esculenta Crantz) is the predominant staple food in Sub-Saharan Africa (SSA) and an industrial crop in South East Asia. Despite focused breeding efforts for increased yield, resistance, and nutritional value, cassava breeding has not advanced at the same rapidity as other staple crops. In the present study, metabolomic techniques were implemented to characterize the chemotypes of selected cassava accessions and assess potential resources for the breeding program. The metabolite data analyzed was applied to describe the biochemical diversity available in the panel, identifying South American accessions as the most diverse. Genotypes with distinct phenotypic traits showed a representative metabolite profile and could be clearly identified, even if the phenotypic trait was a root characteristic, e.g., high amylose content.

A metabolite profiling approach has been implemented to elucidate metabolic adaptation at set culture conditions in five Mycobacterium species (two fast- and three slow-growing) with the potential to act as model organisms for Mycobacterium tuberculosis (Mtb). Analysis has been performed over designated growth phases and under representative environments (nutrient and oxygen depletion) experienced by Mtb during infection. The procedure was useful in determining a range of metabolites (60-120 compounds) covering nucleotides, amino acids, organic acids, saccharides, fatty acids, glycerols, -esters, -phosphates and isoprenoids. Among these classes of compounds, key biomarker metabolites, which can act as indicators of pathway/process activity, were identified. In numerous cases, common metabolite traits were observed for all five species across the experimental conditions (e.g. uracil indicating DNA repair). Amino acid content, especially glutamic acid, highlighted the different properties between the fast- and slow-growing mycobacteria studied (e.g. nitrogen assimilation). The greatest similarities in metabolite composition between fast- and slow-growing mycobacteria were apparent under hypoxic conditions. A comparison to previously reported transcriptomic data revealed a strong correlation between changes in transcription and metabolite content. Collectively, these data validate the changes in the transcription at the metabolite level, suggesting transcription exists as one of the predominant modes of cellular regulation in Mycobacterium. Sectors with restricted correlation between metabolites and transcription (e.g. hypoxic cultivation) warrant further study to elucidate and exploit post-transcriptional modes of regulation. The strong correlation between the laboratory conditions used and data derived from in vivo conditions, indicate that the approach applied is a valuable addition to our understanding of cell regulation in these Mycobacterium species.

In order to decipher the complex biological networks underlying biochemical and physiological processes, cellular regulation at all levels must be studied. The metabolites determined by metabolomics represent the end-point of cellular regulation and thus vital components of any integrative network. In the case of pathogenic agents such as Mycobacterium tuberculosis metabolomics offers an ideal opportunity to gain a better understanding of how this species adapts to environmental conditions and antimicrobial treatments. In the present study a metabolite profiling protocol for Mycobacterium including optimised quenching, extraction and analysis has been devised. These methods have been applied to three different Mycobacterium spp. demonstrating potential translation across the genus. Steady-state levels of metabolites during growth have been determined for Mycobacterium smegmatis, Mycobacterium phlei and Mycobacterium bovis BCG (Bacillus Calmette-Guérin). The changes of designated biomarkers emphasised phenotypical differences (e.g. nitrogen metabolism) and similarities (e.g. cysteine biosynthesis) between the bacteria. Each time point showed distinguishable metabolic characteristics from early lag to late stationary phase/beginning of non-replicating phase. The combination of the metabolic results with published "omics" data indicated that transcription appeared to be the most predominant mode of cellular regulation utilised by these bacteria studied., (Copyright © 2014 Elsevier B.V. All rights reserved.)