Search

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]

• Mbidde bananas show distinct metabolite profile linked to unfavourable sensory properties. • Metabolites associated with sour, sweet, and bitter taste are increased in Mbidde bananas. • Mbidde and Matooke bananas can be defined by distinct metabolites. • Matooke, Mchare and plantains display differential regulation of phenylpropanoid formation. • Metabolomics can contribute to breeding bananas with better consumer quality. Breeding programs for disease resistant bananas in Sub-Saharan Africa generated resistant bananas, which did not meet fruit quality characteristics preferred by consumers. The present study aimed to establish chemotypes underlying preferred cooking bananas of Matooke, Mchare and plantain and less preferred Mbidde bananas, used for beer brewing. The metabolite data of Mbidde showed higher levels of metabolites associated with sour, sweet, and astringent taste; as well as different textural properties associated with cell wall composition and lignin content. Significant differences in the majority of specialised and primary metabolites were observed in the pulp of cooking banana groups. Analysis of peel tissue indicated similar metabolic differences in the protective layer surrounding the pulp and suggested a distinct genetic regulation of phenylpropanoid and flavonoid pathways between the genome groups. In summary, the present data can be used to establish metabolic traits associated with consumer preference, which can augment modern breeding programs. [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]

Cassava (Manihot esculenta Crantz) is an important staple crop for food security in Africa and South America. The present study describes an integrated genomic and metabolomic approach to the characterization of Latin American cassava germplasm. Classification based on genotyping correlated with the leaf metabolome and indicated a key finding of adaption to specific eco-geographical environments. In contrast, the root metabolome did not relate to genotypic clustering, suggesting the different spatial regulation of this tissue's metabolome. The data were used to generate pan-metabolomes for specific tissues, and the inclusion of phenotypic data enabled the identification of metabolic sectors underlying traits of interest. For example, tolerance to whiteflies (Aleurotrachelus socialis) was not linked directly to cyanide content but to cell wall-related phenylpropanoid or apocarotenoid content. Collectively, these data advance the community resources and provide valuable insight into new candidate parental breeding materials with traits of interest directly related to combating food security. [ABSTRACT FROM AUTHOR]

Modern potato breeding programmes have to balance agronomic and consumer preferences, as they seek to develop high yielding and nutritious potatoes that are tolerant to abiotic/biotic stresses. The recent focus on consumer preference has generated sensory data to assess potato flavour intensity, umami, sweetness, sourness, bitterness and mealiness in a panel of 60 cultivated and four wild potato accessions. The potato tubers were assessed immediately after harvest and after a five-week cold storage period. The cultivated accessions included diploid landraces of S. tuberosum L. Andigenum groups Phureja and Stenotonum, diploid hybrids of these, and advanced tetraploid breeding lines. The wild accessions represented tuber-bearing Solanum species, implicated in potato domestication. Sensory data highlighted 20 advanced breeding lines that were relatively weak in potato flavour, sweetness and umami before storage, but increased to the intensities of the landrace and hybrids bred from them after storage. The data observed 39 metabolites which were significantly different between storage times (on average ≥3-fold higher) and associated with sweetness, umami, bitterness and sourness tastes. A comparison to potatoes with high potato flavour emphasised that lower levels of specific metabolites associated with sweetness (e.g. glucose, fructose, glycine and serine), sourness (oxalic, ascorbic and citric acid) and umami (aspartic acid) were related to a higher rating for potato flavour. This corroborates the hypothesis, that perception of sensory traits is based on a complex metabolic composition in potato tubers. These data also indicate that metabolomic analysis can play an important role in augmenting future breeding programmes considering consumer traits. • Wild potatoes scored highest for acidity/bitterness, but the lowest for potato flavour. • Andean landraces had high scores for potato flavour before and after storage. • 20 advanced breeding lines showed improved flavour after storage. • 42 metabolites were associated with changes in sensory evaluation. • Lower levels of specific metabolites were correlated with a higher potato flavour. [ABSTRACT FROM AUTHOR]

Vitamin A deficiency (VAD) in Low and Medium Income countries remains a major health concern. Ipomoea batatas, orange sweet potato (OSP), is one of the biofortification solutions being implemented by the World Health Organisation (WHO) to combat VAD. However, high provitamin A (β-carotene) content has been associated with a reduction in dry matter, reducing calorific value and having adverse effects on consumer traits. Both starch and carotenoid formation are located in amyloplasts and could potentially compete for the same precursors. Hence, five different sweet potato storage root phenotypes were characterized through spatial metabolomics and proteomics at the sub-plastidal level. The metabolite data suggested an indirect correlation of starch and carotenoids through the TCA cycle and pentose phosphate pathway. Furthermore, a change in lipid composition was observed to accommodate the storage of carotenoids in the hydrophilic environment of the amyloplast. The data suggests an alteration of cellular ultra-structures and perturbation of metabolism in high β-carotene producing sweet potato roots. This corroborates with previous gene expression analysis through biochemical analysis of sweet potato root tissue. Interaction between carotenoid and starch biosynthesis in sweet potato amyloplasts. [Display omitted] • Starch and carotenoids compete for the same metabolite precursors. • Reduction of starch is indirectly linked to carotenoids through pyruvate biosynthesis. • Presence of carotenoids changes the cellular ultra-structures of the plastid. • All membrane structures in the root plastid are derived from the plastid envelope. • Functions of membrane structures include synthesis, transport and storage of carotenoids. [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]

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]

Strawberries (Fragaria × ananassa Duch.) are one of the most economically important fruit crops worldwide, several commercially viable cultivars are cultivated in the northern region of Thailand. The morphological characters at the young vegetative seedling stage can be very similar, which has hindered breeding efforts. The present study assesses the ability of random amplification of polymorphic DNA (RAPD) markers and metabolomics techniques to distinguish six strawberry cultivars. Both techniques showed congruent results for the leaf tissue and classified the cultivars into three major clusters. For the most different cultivars, Akihime and Praratchatan No.80, fruits were analysed at eight fruit ripening stages. The data highlighted a broad biological variation at the early ripening stages and less biological variation at the mature stages. Key metabolic differences included the polyphenol profile in Praratchatan No.80 and fatty acid synthesis/oxidation in Akihime. In summary, the RAPD and metabolite data can be used to distinguish strawberry cultivars and elucidate the metabolite composition of each phenotype. This approach to the characterisation of genotypes will benefit future breeding programmes. Image 1 • RAPD markers and metabolite profiles of strawberry leaves showed similar clustering. • Strawberry fruit showed high biological variation at green compared to red-ripe stage. • Fruit metabolism varied between Praratchatan No.80 and Akihime during ripening. • Fatty acid metabolism/catabolism of Akihime results in distinct aroma profile. • Praratchatan No.80 has >2-fold higher levels of polyphenols and flavonoids. [ABSTRACT FROM AUTHOR]

Banana (Musa spp.) plants produce many health promoting compounds in leaf, peel and pulp. For a robust metabolic analysis of these tissues, leaf at five developmental stages were compared to assess suitable sampling practices. Results confirmed that the common sampling practise of leaf 3 is applicable for metabolic comparisons. The developed work flow was applied to analyse the metabolite diversity present in 18 different Musa varieties, providing baseline levels of metabolites in leaf, peel and pulp tissue. Correlation analysis was then used to ascertain whether similar trends can be detected in the three plant tissues of the diversity panel. The genome group displayed a dominant role in the composition of the metabolome in all three tissues. This led to the conclusion that a correlation between tissues was only possible within a genome group as the different parental backgrounds caused too great a variation in the metabolomes. It also suggests the metabolome could be used to monitor the interaction/hybridisation of genomes during breeding programmes. Image 1 • Leaf three is most representative of varietal metabolic composition. • Score plots of leaf, peel and pulp showed common varietal groupings. • Main differences between banana pulps included TCA cycle intermediates and sugars. • Metabolite composition of pulp showed no correlation to consumption type. [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]

• Wild, landrace and bred potato varieties show distinct metabolite profiles. • Group specific metabolite compositions are maintained upon cooking. • Bred native hybrids showed no significant change of Vitamin A precursors. • Accessions with distinct traits were identified for breeding purposes. Elucidating and improving the nutritional value of staple crops is an important focus in breeding programs. Hence, wild and cultivated potatoes have been metabolite profiled to assess metabolic plasticity present in potato tubers and changes that occur in these after cooking. The present data highlighted metabolic similarities of certain wild species to breeding lines, indicating preference during domestication. In the case of potato, the cooking process has to be taken into consideration for the assessment of nutritional quality. The degree of nutritional change during cooking depends on several factors including cooking method, stability of metabolites with nutritional properties and chemotype of the varietal matrix. The differences between raw and boiled tubers were similar within each germplasm group. No notable distinctions of total phenolic levels could be observed within germplasm groups, whereas total carotenoids and glycoalkaloids were significantly decreased. Genotypes/cultivars with notable changes upon cooking distinguished them within their genotype group. [ABSTRACT FROM AUTHOR]

Vitamin A deficiency (VAD) is a global health problem, which despite significant financial investments and initiatives has not been eradicated. Biofortification of staple crops with β-carotene (provitamin A) in Low Medium Income Countries (LMICs) is the approach advocated and adopted by the WHO and HarvestPlus programme. The accurate determination of β-carotene is key to the assessment of outputs from these activities. In the present study, HPLC-PDA analysis displayed superior resolving power, separating and identifying 23 carotenoids in the orange sweet potato (Ipomoea batatas) variety used, including only eight carotenoids with provitamin A properties. Additionally, the results evidently displayed that the use of lyophilised material facilitated the extraction of twice the amount of pigments compared to fresh material, which impacts the precise calculation of the provitamin A content. These results highlight that yellow to orange starchy edible crops produce a wide array of carotenoids in addition to β-carotene. Biosynthetically it is clear from the intermediates and products accumulating that the β-branch of the carotenoid pathway persists in sweet potato tuber material. Collectively, the data also have implications with respect to the determination and biosynthesis of provitamin A among staple crops for developing countries. Image 1 • β-carotene quantification by LC is far more accurate than by spectrophotometer. • 23 different carotenoids are detected by HPLC in orange sweet potato. • Only 9 of 23 carotenoids have provitamin A activity. • Sweet potatoes express a wide range of carotenoid epoxides/furanoids. [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]

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]

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating the OsSBEIIb gene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymeswere up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites. [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]

Key message: Both OsIPPI1 and OsIPPI2 enzymes are found in the endoplasmic reticulum, providing novel important insights into the role of this compartment in the synthesis of MVA pathway isoprenoids. Isoprenoids are synthesized from the precursor's isopentenyl diphosphate (IPP) and dimethylallyl diphosphosphate (DMAPP), which are interconverted by the enzyme isopentenyl diphosphate isomerase (IPPI). Many plants express multiple isoforms of IPPI, the only enzyme shared by the mevalonate (MVA) and non-mevalonate (MEP) pathways, but little is known about their specific roles. Rice (Oryza sativa) has two IPPI isoforms (OsIPPI1 and OsIPPI2). We, therefore, carried out a comprehensive comparison of IPPI gene expression, protein localization, and isoprenoid biosynthesis in this species. We found that OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues, and its expression in de-etiolated leaves mirrored the accumulation of phytosterols, suggesting a key role in the synthesis of MVA pathway isoprenoids. We investigated the subcellular localization of both isoforms by constitutively expressing them as fusions with synthetic green fluorescent protein. Both proteins localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids, due to an N-terminal transit peptide which is not present in OsIPPI1. Despite the plastidial location of OsIPPI2, the expression of OsIPPI2 mRNA did not mirror the accumulation of chlorophylls or carotenoids, indicating that OsIPPI2 may be a redundant component of the MEP pathway. The detection of both OsIPPI isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. Our work shows that the ER plays an as yet unknown role in the synthesis of MVA-derived isoprenoids, with important implications for the metabolic engineering of isoprenoid biosynthesis in higher plants. [ABSTRACT FROM AUTHOR]