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SUMMARY Saffron spice owes its commercial appreciation to its specific apocarotenoids: crocins, picrocrocin, and safranal. In Crocus sativus, these compounds are biosynthesized from zeaxanthin through oxidative cleavage by the carotenoid cleavage dioxygenase 2 (CCD2). Transgenic tomato plants expressing CsCCD2 in the fruit, named Tomaffron, accumulate high levels of saffron apocarotenoids despite the low substrate availability for CsCCD2. In the present study, CsCCD2 has been introduced into Xantomato; this tomato variety accumulates high levels of zeaxanthin and β‐carotene in ripe fruit due to a combination of four mutant alleles. Xantomato and Tomaffron genotypes have been combined to optimize apocarotenoid production. The best transgenic lines accumulated 15 and 14 times more crocins and picrocrocin than Tomaffron, alongside a fourfold increase in β‐carotene compared to Xantomato, albeit at a cost in fruit yield. Segregation of the four mutations has been carried out to find the best combination for obtaining high levels of saffron apocarotenoids without adverse effects on fruit yield. Plants harboring the high‐pigmented 3 (hp3) and BETA (BSh) mutations accumulated 6 and 15 times more crocins and picrocrocin than Tomaffron, without observable pleiotropic effects. Additionally, those high levels of saffron apocarotenoids were obtained in fruit accumulating high levels of both lycopene and β‐carotene independently or in combination, suggesting a regulatory role for the apocarotenoids produced and indicating that it is possible to increase the levels of both types of healthy promoting molecules simultaneously. [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]

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]