Tomato pomace food waste from different variants as a high antioxidant potential resource.

Pomaces obtained from three San Marzano tomato genotypes including the wild type (WT), Sun Black (SB), and colorless fruit epidermis (CL) were dried at 50 °C and analyzed for nutritional composition, total polyphenol (TPC), flavonoid (TFC) content, polyphenol qualitative profile, total antioxidant capacity (TAC), and antimicrobial activity. Commercial dried tomato powder (CTRP) was included as a control. No differences were detected nutritionally, in TPC and antimicrobial activity, but significant changes were observed for TFC and TAC, underlying variation in the phenolic profile. SB pomace (SBP) had the highest TFC and TAC. LC-HRMS analysis showed a flavonoid-enriched profile in SBP besides the exclusive presence of anthocyanins, with petanin and negretein as the most abundant. Among flavonoids, quercetin-hexose-deoxyhexose-pentose, naringenin, and rutin were the major. Overall, we showed the potential of dried tomato pomace, especially SBP, as an extremely valuable waste product to be transformed into a functional ingredient, reducing the food industry waste.

Generation and physiological characterization of genome-edited Nicotiana benthamiana plants containing zeaxanthin as the only leaf xanthophyll.

MAIN CONCLUSION: Simultaneous genome editing of the two homeologous LCYe and ZEP genes of Nicotiana benthamiana results in plants in which all xanthophylls are replaced by zeaxanthin. Plant carotenoids act both as photoreceptors and photoprotectants in photosynthesis and as precursors of apocarotenoids, which include signaling molecules such as abscisic acid (ABA). As dietary components, the xanthophylls lutein and zeaxanthin have photoprotective functions in the human macula. We developed transient and stable combinatorial genome editing methods, followed by direct LC-MS screening for zeaxanthin accumulation, for the simultaneous genome editing of the two homeologous Lycopene Epsilon Cyclase (LCYe) and the two Zeaxanthin Epoxidase (ZEP) genes present in the allopolyploid Nicotiana benthamiana genome. Editing of the four genes resulted in plants in which all leaf xanthophylls were substituted by zeaxanthin, but with different ABA levels and growth habits, depending on the severity of the ZEP1 mutation. In high-zeaxanthin lines, the abundance of the major photosystem II antenna LHCII was reduced with respect to wild-type plants and the LHCII trimeric state became unstable upon thylakoid solubilization. Consistent with the depletion in LHCII, edited plants underwent a compensatory increase in PSII/PSI ratios and a loss of the large-size PSII supercomplexes, while the level of PSI-LHCI supercomplex was unaffected. Reduced activity of the photoprotective mechanism NPQ was shown in high-zeaxanthin plants, while PSII photoinhibition was similar for all genotypes upon exposure to excess light, consistent with the antioxidant and photoprotective role of zeaxanthin in vivo.

A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology.

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor.

Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and ß-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates.

Aglianico Grape Seed Semi-Polar Extract Exerts Anticancer Effects by Modulating MDM2 Expression and Metabolic Pathways.

Grapevine (Vitis vinifera L.) seeds are rich in polyphenols including proanthocyanidins, molecules with a variety of biological effects including anticancer action. We have previously reported that the grape seed semi-polar extract of Aglianico cultivar (AGS) was able to induce apoptosis and decrease cancer properties in different mesothelioma cell lines. Concomitantly, this extract resulted in enriched oligomeric proanthocyanidins which might be involved in determining the anticancer activity. Through transcriptomic and metabolomic analyses, we investigated in detail the anticancer pathway induced by AGS. Transcriptomics analysis and functional annotation allowed the identification of the relevant causative genes involved in the apoptotic induction following AGS treatment. Subsequent biological validation strengthened the hypothesis that MDM2 could be the molecular target of AGS and that it could act in both a p53-dependent and independent manner. Finally, AGS significantly inhibited tumor progression in a xenograft mouse model of mesothelioma, confirming also in vivo that MDM2 could act as molecular player responsible for the AGS antitumor effect. Our findings indicated that AGS, exerting a pro-apoptotic effect by hindering MDM2 pathway, could represent a novel source of anticancer molecules.

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.