Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin.

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using ß-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

Modulation of Caecal Microbiota and Metabolome Profile in Salmonella-Infected Broilers by Phage Therapy.

Bacteriophage therapy is considered one of the most promising tools to control zoonotic bacteria, such as Salmonella, in broiler production. Phages exhibit high specificity for their targeted bacterial hosts, causing minimal disruption to the niche microbiota. However, data on the gut environment's response to phage therapy in poultry are limited. This study investigated the influence of Salmonella phage on host physiology through caecal microbiota and metabolome modulation using high-throughput 16S rRNA gene sequencing and an untargeted metabolomics approach. We employed 24 caecum content samples and 24 blood serum samples from 4-, 5- and 6-week-old broilers from a previous study where Salmonella phages were administered via feed in Salmonella-infected broilers, which were individually weighed weekly. Phage therapy did not affect the alpha or beta diversity of the microbiota. Specifically, we observed changes in the relative abundance of 14 out of the 110 genera using the PLS-DA and Bayes approaches. On the other hand, we noted changes in the caecal metabolites (63 up-accumulated and 37 down-accumulated out of the 1113 caecal metabolites). Nevertheless, the minimal changes in blood serum suggest a non-significant physiological response. The application of Salmonella phages under production conditions modulates the caecal microbiome and metabolome profiles in broilers without impacting the host physiology in terms of growth performance.

A metabolome and transcriptome survey to tap the dynamics of fruit prolonged shelf-life and improved quality within Greek tomato germplasm.

Introduction: Tomato is a high economic value crop worldwide with recognized nutritional properties and diverse postharvest potential. Nowadays, there is an emerging awareness about the exploitation and utilization of underutilized traditional germplasm in modern breeding programs. In this context, the existing diversity among Greek accessions in terms of their postharvest life and nutritional value remains largely unexplored. Methods: Herein, a detailed evaluation of 130 tomato Greek accessions for postharvest and nutritional characteristics was performed, using metabolomics and transcriptomics, leading to the selection of accessions with these interesting traits. Results: The results showed remarkable differences among tomato Greek accessions for overall ripening parameters (color, firmness) and weight loss. On the basis of their postharvest performance, a balance between short shelf life (SSL) and long shelf life (LSL) accessions was revealed. Metabolome analysis performed on 14 selected accessions with contrasting shelf-life potential identified a total of 206 phytonutrients and volatile compounds. In turn, transcriptome analysis in fruits from the best SSL and the best LSL accessions revealed remarkable differences in the expression profiles of transcripts involved in key metabolic pathways related to fruit quality and postharvest potential. Discussion: The pathways towards cell wall synthesis, polyamine synthesis, ABA catabolism, and steroidal alkaloids synthesis were mostly induced in the LSL accession, whereas those related to ethylene biosynthesis, cell wall degradation, isoprenoids, phenylpropanoids, ascorbic acid and aroma (TomloxC) were stimulated in the SSL accession. Overall, these data would provide valuable insights into the molecular mechanism towards enhancing shelf-life and improving flavor and aroma of modern tomato cultivars.

Deregulation of ζ-carotene desaturase in Arabidopsis and tomato exposes a unique carotenoid-derived redundant regulation of floral meristem identity and function.

A level of redundancy and interplay among the transcriptional regulators of floral development safeguards a plant's reproductive success and ensures crop production. In the present study, an additional layer of complexity in the regulation of floral meristem (FM) identity and flower development is elucidated linking carotenoid biosynthesis and metabolism to the regulation of determinate flowering. The accumulation and subsequent cleavage of a diverse array of ζ-carotenes in the chloroplast biogenesis 5 (clb5) mutant of Arabidopsis results in the reprogramming of meristematic gene regulatory networks establishing FM identity mirroring that of the FM identity master regulator, APETALA1 (AP1). The immediate transition to floral development in clb5 requires long photoperiods in a GIGANTEA-independent manner, whereas AP1 is essential for the floral organ development of clb5. The elucidation of this link between carotenoid metabolism and floral development translates to tomato exposing a regulation of FM identity redundant to and initiated by AP1 and proposed to be dependent on the E class floral initiation and organ identity regulator, SEPALLATA3 (SEP3).

A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota.

Plants generate energy flows through natural food webs, driven by competition for resources among organisms, which are part of a complex network of multitrophic interactions. Here, we demonstrate that the interaction between tomato plants and a phytophagous insect is driven by a hidden interplay between their respective microbiotas. Tomato plants colonized by the soil fungus Trichoderma afroharzianum, a beneficial microorganism widely used in agriculture as a biocontrol agent, negatively affects the development and survival of the lepidopteran pest Spodoptera littoralis by altering the larval gut microbiota and its nutritional support to the host. Indeed, experiments aimed to restore the functional microbial community in the gut allow a complete rescue. Our results shed light on a novel role played by a soil microorganism in the modulation of plant-insect interaction, setting the stage for a more comprehensive analysis of the impact that biocontrol agents may have on ecological sustainability of agricultural systems.

Biosynthesis of Chlorophyll and Other Isoprenoids in the Plastid of Red Grape Berry Skins.

Despite current knowledge showing that fruits like tomato and grape berries accumulate different components of the light reactions and Calvin cycle, the role of green tissues in fruits is not yet fully understood. In mature tomato fruits, chlorophylls are degraded and replaced by carotenoids through the conversion of chloroplasts in chromoplasts, while in red grape berries, chloroplasts persist at maturity and chlorophylls are masked by anthocyanins. To study isoprenoid and lipid metabolism in grape skin chloroplasts, metabolites of enriched organelle fractions were analyzed by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) and the expression of key genes was evaluated by real-time polymerase chain reaction (PCR) in berry skins and leaves. Overall, the results indicated that chloroplasts of the grape berry skins, as with leaf chloroplasts, share conserved mechanisms of synthesis (and degradation) of important components of the photosynthetic machinery. Some of these components, such as chlorophylls and their precursors, and catabolites, carotenoids, quinones, and lipids have important roles in grape and wine sensory characteristics.

A xanthophyll-derived apocarotenoid regulates carotenogenesis in tomato chromoplasts.

Carotenoids possess important biological functions that make them essential components of the human diet. ß-Carotene and some other carotenoids have vitamin A activity while lutein and zeaxanthin, typically referred to as the macular pigments, are involved in good vision and in delaying the onset of age-related eye diseases. In order to create a zeaxanthin-producing tomato fruit, two transgenic lines, one with a high ß-carotene cyclase activity and the other with a high ß-carotene hydroxylase activity, have been genetically crossed. Ripe fruits from the resulting progeny contained significant levels of violaxanthin, antheraxanthin, and xanthophyll esters. However, their zeaxanthin content was not as high as expected, and the total level of carotenoids was only 25% of the carotenoids found in ripe fruits of the comparator line. Targeted transcript analysis and apocarotenoids determinations indicated that transcriptional regulation of the pathway or degradation of synthesized carotenoids were not responsible for the low carotenoid content of hybrid fruits which instead appeared to result from a substantial reduction of carotenoid biosynthesis. Notably, the content of an unidentified hydroxylated cyclic (C13) apocarotenoid was 13 times higher in the hybrid fruits than in the control fruits. Furthermore, a GC-MS-based metabolite profiling demonstrated a perturbation of carotenogenesis in ripening hybrid fruits compatible with a block of the pathway. Moreover, carotenoid profiling on leaf, fruit, and petal samples from a set of experimental lines carrying the hp3 mutation, in combination with the two transgenes, indicated that the carotenoid biosynthesis in petal and fruit chromoplasts could be regulated. Altogether the data were consistent with the hypothesis of the regulation of the carotenoid pathway in tomato chromoplasts through a mechanism of feedback inhibition mediated by a xanthophyll-derived apocarotenoid. This chromoplast-specific post-transcriptional mechanism was disclosed in transgenic fruits of HU hybrid owing to the abnormal production of zeaxanthin and antheraxanthin, the more probable precursors of the apocarotenoid signal. A model describing the regulation of carotenoid pathway in tomato chromoplasts is presented.

Fortification and bioaccessibility of saffron apocarotenoids in potato tubers.

Carotenoids are C40 isoprenoids with well-established roles in photosynthesis, pollination, photoprotection, and hormone biosynthesis. The enzymatic or ROS-induced cleavage of carotenoids generates a group of compounds named apocarotenoids, with an increasing interest by virtue of their metabolic, physiological, and ecological activities. Both classes are used industrially in a variety of fields as colorants, supplements, and bio-actives. Crocins and picrocrocin, two saffron apocarotenoids, are examples of high-value pigments utilized in the food, feed, and pharmaceutical industries. In this study, a unique construct was achieved, namely O6, which contains CsCCD2L, UGT74AD1, and UGT709G1 genes responsible for the biosynthesis of saffron apocarotenoids driven by a patatin promoter for the generation of potato tubers producing crocins and picrocrocin. Different tuber potatoes accumulated crocins and picrocrocin ranging from 19.41-360 to 105-800 µg/g DW, respectively, with crocetin, crocin 1 [(crocetin-(ß-D-glucosyl)-ester)] and crocin 2 [(crocetin)-(ß-D-glucosyl)-(ß-D-glucosyl)-ester)] being the main compounds detected. The pattern of carotenoids and apocarotenoids were distinct between wild type and transgenic tubers and were related to changes in the expression of the pathway genes, especially from PSY2, CCD1, and CCD4. In addition, the engineered tubers showed higher antioxidant capacity, up to almost 4-fold more than the wild type, which is a promising sign for the potential health advantages of these lines. In order to better investigate these aspects, different cooking methods were applied, and each process displayed a significant impact on the retention of apocarotenoids. More in detail, the in vitro bioaccessibility of these metabolites was found to be higher in boiled potatoes (97.23%) compared to raw, baked, and fried ones (80.97, 78.96, and 76.18%, respectively). Overall, this work shows that potatoes can be engineered to accumulate saffron apocarotenoids that, when consumed, can potentially offer better health benefits. Moreover, the high bioaccessibility of these compounds revealed that potato is an excellent way to deliver crocins and picrocrocin, while also helping to improve its nutritional value.

Examining the effects of Salmonella phage on the caecal microbiota and metabolome features in Salmonella-free broilers.

Bacteriophages selectively infect and kill their target bacterial host, being a promising approach to controlling zoonotic bacteria in poultry production. To ensure confidence in its use, fundamental questions of safety and toxicity monitoring of phage therapy should be raised. Due to its high specificity, a minimal impact on the gut ecology is expected; however, more in-depth research into key parameters that influence the success of phage interventions has been needed to reach a consensus on the impact of bacteriophage therapy in the gut. In this context, this study aimed to investigate the interaction of phages with animals; more specifically, we compared the caecum microbiome and metabolome after a Salmonella phage challenge in Salmonella-free broilers, evaluating the role of the phage administration route. To this end, we employed 45 caecum content samples from a previous study where Salmonella phages were administered via drinking water or feed for 24 h from 4, 5 to 6-weeks-old broilers. High-throughput 16S rRNA gene sequencing showed a high level of similarity (beta diversity) but revealed a significant change in alpha diversity between broilers with Salmonella-phage administered in the drinking water and control. Our results showed that the phages affected only a few genera of the microbiota's structure, regardless of the administration route. Among these, we found a significant increase in Streptococcus and Sellimonas in the drinking water and Lactobacillus, Anaeroplasma and Clostridia_vadinBB60_group in the feed. Nevertheless, the LC-HRMS-based metabolomics analyses revealed that despite few genera were significantly affected, a substantial number of metabolites, especially in the phage administered in the drinking water were significantly altered (64 and 14 in the drinking water and feed groups, respectively). Overall, our study shows that preventive therapy with bacteriophages minimally alters the caecal microbiota but significantly impacts their metabolites, regardless of the route of administration.

Gut-Brain Axis: Insights from Hippocampal Neurogenesis and Brain Tumor Development in a Mouse Model of Experimental Colitis Induced by Dextran Sodium Sulfate.

Chronic inflammatory bowel disorders (IBD) are idiopathic diseases associated with altered intestinal permeability, which in turn causes an exaggerated immune response to enteric antigens in a genetically susceptible host. A rise in psych cognitive disorders, such as anxiety and depression, has been observed in IBD patients. We here report investigations on a model of chemically induced experimental colitis by oral administration of sodium dextran sulfate (DSS) in C57BL/6 mice. We investigate, in vivo, the crosstalk between the intestine and the brain, evaluating the consequences of intestinal inflammation on neuroinflammation and hippocampal adult neurogenesis. By using different DSS administration strategies, we are able to induce acute or chronic colitis, simulating clinical characteristics observed in IBD patients. Body weight loss, colon shortening, alterations of the intestinal mucosa and fecal metabolic changes in amino acids-, lipid- and thiamine-related pathways are observed in colitis. The activation of inflammatory processes in the colon is confirmed by macrophage infiltration and increased expression of the proinflammatory cytokine and oxidative stress marker (Il-6 and iNOS). Interestingly, in the hippocampus of acutely DSS-treated mice, we report the upregulation of inflammatory-related genes (Il-6, Il-1ß, S-100, Tgf-ß and Smad-3), together with microgliosis. Chronic DSS treatment also resulted in neuroinflammation in the hippocampus, indicated by astrocyte activation. Evaluation of stage-specific neurogenesis markers reveals deficits in the dentate gyrus after acute and chronic DSS treatments, indicative of defective adult hippocampal neurogenesis. Finally, based on a possible causal relationship between gut-related inflammation and brain cancer, we investigate the impact of DSS-induced colitis on oncogenesis, using the Ptch1+/-/C57BL/6 mice, a well-established medulloblastoma (MB) mouse model, finding no differences in MB development between untreated and DSS-treated mice. In conclusion, in our experimental model, the intestinal inflammation associated with acute and chronic colitis markedly influences brain homeostasis, impairing hippocampal neurogenesis but not MB oncogenesis.

Engineering high levels of saffron apocarotenoids in tomato.

Crocins and picrocrocin are high-value hydrophilic pigments produced in saffron and used commercially in the food and pharmaceutical industries. These apocarotenoids are derived from the oxidative cleavage of zeaxanthin by specific carotenoid cleavage dioxygenases. The pathway for crocins and picrocrocin biosynthesis was introduced into tomato using fruit specific and constitutive promoters and resulted in 14.48 mg/g of crocins and 2.92 mg/g of picrocrocin in the tomato DW, without compromising plant growth. The strategy involved expression of CsCCD2L to produce crocetin dialdehyde and 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene, and of glycosyltransferases UGT709G1 and CsUGT2 for picrocrocin and crocins production, respectively. Metabolic analyses of the engineered fruits revealed picrocrocin and crocetin-(ß-D-gentiobiosyl)-(ß-D-glucosyl)-ester, as the predominant crocin molecule, as well as safranal, at the expense of the usual tomato carotenoids. The results showed the highest crocins content ever obtained by metabolic engineering in heterologous systems. In addition, the engineered tomatoes showed higher antioxidant capacity and were able to protect against neurological disorders in a Caenorhabditis elegans model of Alzheimer's disease. Therefore, these new developed tomatoes could be exploited as a new platform to produce economically competitive saffron apocarotenoids with health-promoting properties.

Heterologous expression of Bixa orellana cleavage dioxygenase 4-3 drives crocin but not bixin biosynthesis.

Annatto (Bixa orellana) is a perennial shrub native to the Americas, and bixin, derived from its seeds, is a methoxylated apocarotenoid used as a food and cosmetic colorant. Two previous reports claimed to have isolated the carotenoid cleavage dioxygenase (CCD) responsible for the production of the putative precursor of bixin, the C24 apocarotenal bixin dialdehyde. We re-assessed the activity of six Bixa CCDs and found that none of them produced substantial amounts of bixin dialdehyde in Escherichia coli. Unexpectedly, BoCCD4-3 cleaved different carotenoids (lycopene, ß-carotene, and zeaxanthin) to yield the C20 apocarotenal crocetin dialdehyde, the known precursor of crocins, which are glycosylated apocarotenoids accumulated in saffron stigmas. BoCCD4-3 lacks a recognizable transit peptide but localized to plastids, the main site of carotenoid accumulation in plant cells. Expression of BoCCD4-3 in Nicotiana benthamiana leaves (transient expression), tobacco (Nicotiana tabacum) leaves (chloroplast transformation, under the control of a synthetic riboswitch), and in conjunction with a saffron crocetin glycosyl transferase, in tomato (Solanum lycopersicum) fruits (nuclear transformation) led to high levels of crocin accumulation, reaching the highest levels (>100 µg/g dry weight) in tomato fruits, which also showed a crocin profile similar to that found in saffron, with highly glycosylated crocins as major compounds. Thus, while the bixin biosynthesis pathway remains unresolved, BoCCD4-3 can be used for the metabolic engineering of crocins in a wide range of different plant tissues.

Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit.

Embryo manipulation is a requisite step in assisted reproductive technology (ART). Therefore, it is of great necessity to appraise the safety of ART and investigate the long-term effect, including lipid metabolism, on ART-conceived offspring. Augmenting our ART rabbit model to investigate lipid metabolic outcomes in offspring longitudinally, we detected variations in hepatic DNA methylation ART offspring in the F3 generation for embryonic exposure (multiple ovulation, vitrification and embryo transfer). Through adult liver metabolomics and proteomics, we identified changes mainly related to lipid metabolism (e.g., polyunsaturated fatty acids, steroids, steroid hormone). We also found that DNA methylation analysis was linked to changes in lipid metabolism and apoptosis genes. Nevertheless, these differences did not apparently alter the general health status. Thus, our findings suggest that ART is likely to be a player in embryo epigenetic events related to hepatic homeostasis alteration in adulthood.

A New Glycosyltransferase Enzyme from Family 91, UGT91P3, Is Responsible for the Final Glucosylation Step of Crocins in Saffron (Crocus sativus L.).

Crocetin is an apocarotenoid formed from the oxidative cleavage of zeaxanthin, by the carotenoid cleavage enzymes CCD2 (in Crocus species) and specific CCD4 enzymes in Buddleja davidii and Gardenia jasminoides. Crocetin accumulates in the stigma of saffron in the form of glucosides and crocins, which contain one to five glucose molecules. Crocetin glycosylation was hypothesized to involve at least two enzymes from superfamily 1 UDP-sugar dependent glycosyltransferases. One of them, UGT74AD1, produces crocins with one and two glucose molecules, which are substrates for a second UGT, which could belong to the UGT79, 91, or 94 families. An in silico search of Crocus transcriptomes revealed six candidate UGT genes from family 91. The transcript profiles of one of them, UGT91P3, matched the metabolite profile of crocin accumulation, and were co-expressed with UGT74AD1. In addition, both UGTs interact in a two-hybrid assay. Recombinant UGT91P3 produced mostly crocins with four and five glucose molecules in vitro, and in a combined transient expression assay with CCD2 and UGT74AD1 enzymes in Nicotiana benthamiana. These results suggest a role of UGT91P3 in the biosynthesis of highly glucosylated crocins in saffron, and that it represents the last missing gene in crocins biosynthesis.

Identification and characterization of apocarotenoid modifiers and carotenogenic enzymes for biosynthesis of crocins in Buddleja davidii flowers.

Crocetin biosynthesis in Buddleja davidii flowers proceeds through a zeaxanthin cleavage pathway catalyzed by two carotenoid cleavage dioxygenases (BdCCD4.1 and BdCCD4.3), followed by oxidation and glucosylation reactions that lead to the production of crocins. We isolated and analyzed the expression of 12 genes from the carotenoid pathway in B. davidii flowers and identified four candidate genes involved in the biosynthesis of crocins (BdALDH, BdUGT74BC1, BdUGT74BC2, and BdUGT94AA3). In addition, we characterized the profile of crocins and their carotenoid precursors, following their accumulation during flower development. Overall, seven different crocins, crocetin, and picrocrocin were identified in this study. The accumulation of these apocarotenoids parallels tissue development, reaching the highest concentration when the flower is fully open. Notably, the pathway was regulated mainly at the transcript level, with expression patterns of a large group of carotenoid precursor and apocarotenoid genes (BdPSY2, BdPDS2, BdZDS, BdLCY2, BdBCH, BdALDH, and BdUGT Genes) mimicking the accumulation of crocins. Finally, we used comparative correlation network analysis to study how the synthesis of these valuable apocarotenoids diverges among B. davidii, Gardenia jasminoides, and Crocus sativus, highlighting distinctive differences which could be the basis of the differential accumulation of crocins in the three species.

VviRafS5 Is a Raffinose Synthase Involved in Cold Acclimation in Grapevine Woody Tissues.

The accumulation of raffinose family oligosaccharides (RFOs) is a hallmark of plant response to different abiotic stresses, including cold. The synthesis of galactinol, by galactinol synthases (GolS), and raffinose, by raffinose synthases (RafS), are fundamental for stress-induced accumulation of RFOs, but the role of these enzymes in the cold response of grapevine (Vitis vinifera L.) woody tissues is still unclear. To address this gap in the literature, 1-year-lignified grapevine canes were incubated at 4°C for 7 and 14 days and tissues were analyzed for sugar content and gene expression. Results showed that, in parallel to starch breakdown, there was an increase in soluble sugars, including sucrose, glucose, fructose, raffinose, and stachyose. Remarkably, abscisic acid (ABA) levels increased during cold acclimation, which correlated with the increased expression of the key ABA-synthesis genes VviNCED2 and VviNCED3. Expression analysis of the VviGolS and VviRafS family allowed the identification of VviRafS5 as a key player in grapevine cold response. The overexpression of VviRafS5 in Saccharomyces cerevisiae allowed the biochemical characterization of the encoded protein as a raffinose synthase with a size of ~87 kDa. In grapevine cultured cells, VviRafS5 was upregulated by cold and ABA but not by heat and salt stresses. Our results suggest that ABA accumulation in woody tissues during cold acclimation upregulates VivRafS5 leading to raffinose synthesis.

Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer.

Although assisted reproduction technologies (ARTs) are recognised as safe, and most of the offspring seem apparently healthy, there is clear evidence that ARTs are associated with changes in the embryo's developmental trajectory, which incur physiological consequences during the prenatal and postnatal stages of life. The present study aimed to address the influence of early (day-3 embryos) embryo transfer and cryopreservation on embryo survival, size, and metabolome at the preimplantation stage (day-6 embryos). To this end, fresh-transferred (FT) and vitrified-transferred (VT) embryos were compared using naturally-conceived (NC) embryos as a control reference. The results show that as in vitro manipulation was increased (NC < FT < VT), both embryo survival rate (0.91 ± 0.02, 0.78 ± 0.05 and 0.63 ± 0.05, for NC, FT, and VT groups, respectively) and embryo size (3.21 ± 0.49 mm, 2.15 ± 0.51 mm, 1.76 ± 0.46 mm of diameter for NC, FT, and VT groups, respectively) were significantly decreased. Moreover, an unbiased metabolomics analysis showed overall down-accumulation in 40 metabolites among the three experimental groups, with embryo transfer and embryo cryopreservation procedures both exerting a cumulative effect. In this regard, targeted metabolomics findings revealed a significant reduction in some metabolites involved in metabolic pathways, such as the Krebs cycle, amino acids, unsaturated fatty acids, and arachidonic acid metabolisms. Altogether, these findings highlight a synergistic effect between the embryo transfer and vitrification procedures in preimplantation embryos. However, the ex vivo manipulation during embryo transfer seemed to be the major trigger of the embryonic changes, as the deviations added by the vitrification process were relatively smaller.

Flavescence Dorée-Derived Leaf Yellowing in Grapevine (Vitis vinifera L.) Is Associated to a General Repression of Isoprenoid Biosynthetic Pathways.

Flavescence dorée (FD), caused by the phytoplasma Candidatus Phytoplasma vitis, is a major threat to vineyard survival in different European grape-growing areas. It has been recorded in French vineyards since the mid-1950s, and rapidly spread to other countries. In Portugal, the phytoplasma was first detected in the DOC region of 'Vinhos Verdes' in 2006, and reached the central region of the country in 2009. The infection causes strong accumulation of carbohydrates and phenolics in the mesophyll cells and a simultaneous decrease of chlorophylls, events accompanied by a down regulation of genes and proteins involved in the dark and light-dependent reactions and stabilization of the photosystem II (PSII). In the present study, to better elucidate the basis of the leaf chlorosis in infected grapevine cv. Loureiro, we studied the isoprenoid transcript-metabolite correlation in leaves from healthy and FD-infected vines. Specifically, targeted metabolome revealed that twenty-one compounds (out of thirty-two), including chlorophylls, carotenoids, quinones and tocopherols, were reduced in response to FD-infection. Thereafter, and consistently with the biochemical data, qPCR analysis highlighted a severe FD-mediated repression in key genes involved in isoprenoid biosynthetic pathways. A more diverse set of changes, on the contrary, was observed in the case of ABA metabolism. Principal component analysis (PCA) of all identified metabolites clearly separated healthy from FD-infected vines, therefore confirming that the infection strongly alters the biosynthesis of grapevine isoprenoids; additionally, forty-four genes and metabolites were identified as the components mostly explaining the variance between healthy and infected samples. Finally, transcript-metabolite network correlation analyses were exploited to display the main hubs of the infection process, which highlighted a strong role of VvCHLG, VvVTE and VvZEP genes and the chlorophylls intermediates aminolevulunic acid and porphobilinogen in response to FD infection. Overall, results indicated that the FD infection impairs the synthesis of isoprenoids, through the repression of key genes involved in the biosynthesis of chlorophylls, carotenoids, quinones and tocopherols.

Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system.

Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, for the color and the unique taste of the saffron spice, known as red gold due to its high price. Several studies have also shown the health-promoting properties of these compounds. However, their high costs hamper the wide use of these metabolites in the pharmaceutical sector. We have developed a virus-driven system to produce remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants in only two weeks. The system consists of viral clones derived from tobacco etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that the sole virus-driven expression of C. sativus CsCCD2L or B. davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. In an attempt to improve apocarotenoid content in N. benthamiana, co-expression of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene synthase (PaCrtB) and saffron ß-carotene hydroxylase 2 (BCH2), was performed using the same viral system. This combinatorial approach led to an additional crocin increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. Considering that saffron apocarotenoids are costly harvested from flower stigma once a year, and that Buddleja spp. flowers accumulate lower amounts, this system may be an attractive alternative for the sustainable production of these appreciated metabolites.

Color Mutations Alter the Biochemical Composition in the San Marzano Tomato Fruit.

San Marzano (SM) is a traditional Italian landrace characterized by red elongated fruits, originating in the province of Naples (Italy) and cultivated worldwide. Three mutations, yellow flesh (r), green flesh (gf) and colorless fruit epidermis (y) were introduced into SM by backcross and the resulting introgression lines (ILs) produced the expected yellow, brown and pink fruit variants. In addition, ILs carrying double combinations of those mutations were obtained. The six ILs plus the SM reference were analyzed for volatile (VOC), non-polar (NP) and polar (P) metabolites. Sixty-eight VOCs were identified, and several differences evidenced in the ILs; overall gf showed epistasis over r and y and r over y. Analysis of the NP component identified 54 metabolites; variation in early carotenoids (up to lycopene) and chlorophylls characterized respectively the ILs containing r and gf. In addition, compounds belonging to the quinone and xanthophyll classes were present in genotypes carrying the r mutation at levels higher than SM. Finally, the analysis of 129 P metabolites evidenced different levels of vitamins, amino acids, lipids and phenylpropanoids in the ILs. A correlation network approach was used to investigate metabolite-metabolite relationships in the mutant lines. Altogether these differences potentially modified the hedonistic and nutritional value of the berry. In summary, single and combined mutations in gf, r and y generated interesting visual and compositional diversity in the SM landrace, while maintaining its original typology.