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Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 52 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered 7 novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae, and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity.

Crocins are high-value water soluble pigments that have long been recognized for their medicinal properties, and whose demand has increased worldwide in recent years. In the present study transgenic tomato fruits engineered for the production of crocins and hp3/Bsh tomato mutants with alterations in the carotenoid metabolism, have been combined to increase the levels of crocins in tomato fruits. Tomato fruits from F4 plants showed high antioxidant capacity and the crocin-rich juice of the produced tomato fruit was protected neuroblastoma cells against oxidative insult, through its ability to activate factor 2 related to nuclear erythroid factor 2 (Nrf2). The bio-accessibility of crocins in the juice showed values similar to the ones observed with saffron. Overall, our results support that crocin-fortified tomatoes could result in higher crocin availability and have additional health-promoting effects and could provide better protection against oxidative stress related chronic diseases in humans.

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-(β-D-glucosyl)-ester)] and crocin 2 [(crocetin)-(β-D-glucosyl)-(β-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.

Crocins are high-value soluble pigments that are used as colorants and supplements, their presence in nature is extremely limited and, consequently, the high cost of these metabolites hinders their use by other sectors, such as the pharmaceutical and cosmetic industries. The carotenoid cleavage dioxygenase 2L (CsCCD2L) is the key enzyme in the biosynthetic pathway of crocins in Crocus sativus. In this study, CsCCD2L was introduced into Nicotiana tabacum and Nicotiana glauca for the production of crocins. In addition, a chimeric construct containing the Brevundimonas sp. β-carotene hydroxylase (BrCrtZ), the Arabidopsis thaliana ORANGE mutant gene (AtOrMut), and CsCCD2L was also introduced into N. tabacum. Quantitative and qualitative studies on carotenoids and apocarotenoids in the transgenic plants expressing CsCCD2L alone showed higher crocin level accumulation in N. glauca transgenic plants, reaching almost 400 μg/g DW in leaves, while in N. tabacum 36 μg/g DW was obtained. In contrast, N. tabacum plants coexpressing CsCCD2L, BrCrtZ, and AtOrMut accumulated, 3.5-fold compared to N. tabacum plants only expressing CsCCD2L. Crocins with three and four sugar molecules were the main molecular species in both host systems. Our results demonstrate that the production of saffron apocarotenoids is feasible in engineered Nicotiana species and establishes a basis for the development of strategies that may ultimately lead to the commercial exploitation of these valuable pigments for multiple applications.

[Display omitted] • Crocins are hydrophilic, yellow apocarotenoids produced by a few plant species including saffron. • Crocins have been used by humans for centuries due to their pigment and health properties. • A feasible and economic production of crocins can be obtained using a combination of tomato engineered plants and mutants. • The crocins from the tomato extracts are highly bioaccessible and shown antioxidant activities. Crocins are high-value water soluble pigments that have long been recognized for their medicinal properties, and whose demand has increased worldwide in recent years. In the present study transgenic tomato fruits engineered for the production of crocins and hp3/Bsh tomato mutants with alterations in the carotenoid metabolism, have been combined to increase the levels of crocins in tomato fruits. Tomato fruits from F 4 plants showed high antioxidant capacity and the crocin-rich juice of the produced tomato fruit was protected neuroblastoma cells against oxidative insult, through its ability to activate factor 2 related to nuclear erythroid factor 2 (Nrf2). The bio-accessibility of crocins in the juice showed values similar to the ones observed with saffron. Overall, our results support that crocin-fortified tomatoes could result in higher crocin availability and have additional health-promoting effects and could provide better protection against oxidative stress related chronic diseases in humans. [ABSTRACT FROM AUTHOR]

• The apocarotenoid crocetin and its glycosylated derivatives, crocins, confer the red colour to saffron. Crocetin biosynthesis in saffron is catalysed by the carotenoid cleavage dioxygenase CCD2 (AIG94929). No homologues have been identified in other plant species due to the very limited presence of crocetin and its derivatives in the plant kingdom. • Spring Crocus species with yellow flowers accumulate crocins in the stigma and tepals. Four carotenoid CCDs, namely CaCCD1, CaCCD2 and CaCCD4a/b and CaCCD4c were first cloned and characterized. • CaCCD2 was localized in plastids, and a longer CCD2 version, CsCCD2L, was also localized in this compartment. The activity of CaCCD2 was assessed in Escherichia coli and in a stable rice gene function characterization system, demonstrating the production of crocetin in both systems. The expression of all isolated CCDs was evaluated in stigma and tepals at three key developmental stages in relation with apocarotenoid accumulation. CaCCD2 expression parallels crocin accumulation, but C14 apocarotenoids most likely are associated to the CaCCD1 activity in Crocus ancyrensis flowers. • The specific CCD2 localization and its membrane interaction will contribute to the development of a better understanding of the mechanism of crocetin biosynthesis and regulation in the chromoplast. [ABSTRACT FROM AUTHOR]

Glycosyltransferases play diverse roles in cellular metabolism by modifying the activities of regulatory metabolites. Three stress-regulated UDP-glucosyltransferase-encoding genes have been isolated from the stigmas of saffron, UGT85U1, UGT85U2 and UGT85V1, which belong to the UGT85 family that includes members associated with stress responses and cell cycle regulation. Arabidopsis constitutively expressing UGT85U1 exhibited and increased anchor root development. No differences were observed in the timing of root emergence, in leaf, stem and flower morphology or flowering time. However, salt and oxidative stress tolerance was enhanced in these plants. Levels of glycosylated compounds were measured in these plants and showed changes in the composition of several indole-derivatives. Moreover, auxin levels in the roots were higher compared to wild type. The expression of several key genes related to root development and auxin homeostasis, including CDKB2.1 , CDKB2.2 , PIN2 , 3 and 4 ; TIR1 , SHR , and CYCD6 , were differentially regulated with an increase of expression level of SHR , CYCD6 , CDKB2.1 and PIN2 . The obtained results showed that UGT85U1 takes part in root growth regulation via auxin signal alteration and the modified expression of cell cycle-related genes, resulting in significantly improved survival during oxidative and salt stress treatments. [ABSTRACT FROM AUTHOR]

Background In saffron (Crocus sativus), new corms develop at the base of every shoot developed from the maternal corm, a globular underground storage stem. Since the degree of bud sprouts influences the number and size of new corms, and strigolactones (SLs) suppress growth of pre-formed axillary bud, it was considered appropriate to investigate SL involvement in physiology and molecular biology in saffron. We focused on two of the genes within the SL pathway, CCD7 and CCD8, encoding carotenoid cleavage enzymes required for the production of SLs. Results The CsCCD7 and CsCCD8 genes are the first ones isolated and characterized from a nongrass monocotyledonous plant. CsCCD7 and CsCCD8 expression showed some overlapping, although they were not identical. CsCCD8 was highly expressed in quiescent axillary buds and decapitation dramatically reduced its expression levels, suggesting its involvement in the suppression of axillary bud outgrowth. Furthermore, in vitro experiments showed also the involvement of auxin, cytokinin and jasmonic acid on the sprouting of axillary buds from corms in which the apical bud was removed. In addition, CsCCD8 expression, but not CsCCD7, was higher in the newly developed vascular tissue of axillary buds compared to the vascular tissue of the apical bud. Conclusions We showed that production and transport of auxin in saffron corms could act synergistically with SLs to arrest the outgrowth of the axillary buds, similar to the control of above-ground shoot branching. In addition, jasmonic acid seems to play a prominent role in bud dormancy in saffron. While cytokinins from roots promote bud outgrowth. In addition the expression results of CsCCD8 suggest that SLs could positively regulate procambial activity and the development of new vascular tissues connecting leaves with the mother corm. [ABSTRACT FROM AUTHOR]

Highlights: [•] The external (ECA) and internal (ICA) area of saffron corm were lyophilized and sterilized to prepare agar plates. [•] ECA showed the highest inhibitory effects against the five isolated fungi from infected corms during August. [•] The saponin fraction exclusively detected on ECA, is partially responsible for its extra effect. [•] The main inhibitory effect of these saponins was against Fusarium oxysporum followed by Bipolaris spicifera and Aspergillus niger. [•] The inhibitory effect against Penicillium raistriicki and Rhizopus nigricans was almost negligent. [ABSTRACT FROM AUTHOR]

Abstract: A mixture of highly glycosilated triterpenoid saponins (CS5) isolated from the corm of Crocus sativus or saffron showed cytotoxic activity against HeLa tumoral cells. The main reverse phase HPLC fraction of this mixture (CS5-1) contains two new oleanane-type saponins, denominated Azafrine 1 (1) and Azafrine 2 (2). The bidesmosidic saponins were respectively characterized as (1) 3-O-β-d-glucopyranosiduronic acid echinocystic acid 28-O-β-D-galactopyranosyl-(1→2)-α-l-arabinopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→4)]-α-d-rhamnopyranosyl-(1→2)-[4-O-di-α-L-rhamnopyranosyl-3,16-dihydroxy-10-oxo-hexadecanoyl]-α-D-fucopyranoside and (2) 3-O-β-D-galactopyranosiduronic acid echinocystic acid 28-O-β-D-galactopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)-[4-O-di-α-L-rhamnopyranosyl-3,16-dihydroxy-10-oxo-hexadecanoyl]-β-D-fucopyranoside. The surfactant properties of saponins, probably involved in the cytotoxic activity of CS5 and their exclusive localization in the external part or the corm, indicate their possible role as phytoprotectans. The similarity of their structural compositions to that of other triterpenoid saponins which are of special use in the pharmaceutical industry suggest a new application for C. sativus crops through the exploitation of corm for saponin extraction. [Copyright &y& Elsevier]

Abstract: Saffron, the desiccated stigmas of Crocus sativus, is highly appreciated by its peculiar colour, flavour and aroma. The main compounds that accumulated throughout stigma development in C. sativus are crocetin, its glucoside derivatives, crocins, and picrocrocin, all of which increased as stigmas reached a fully developed stage. After anthesis, and in the absence of fertilization, the flower enters in a senescence programme, which represents the ultimate stage of floral development and results in wilting of whole flower. The programmed senescence of flowers allows the removal of a metabolically active tissue. We studied the composition of saffron apocarotenoids during the senescence of C. sativus flowers, and observed that changes in crocins were due to their transport from the senescent stigma to the ovaries and the developing corm. Afterwards, deglucosylation of crocins in these tissues results in crocetin accumulation. This mobilization mimics the export to storage cells (resorbed) of different compounds during leaf senescence avoiding loss of nutrients in leaves that would otherwise be cycled back into the soil system through leaf litter decomposition. In C. sativus, the resorbed apocarotenoids are stored within the developing corm, where they are not further detected in the advanced stages of development, suggesting that they are metabolized during the early and active phases of corm development, where the glucose molecules from crocins might contribute to cell initiation and elongation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In saffron, the cleavage of zeaxanthin by means of CCD2 generates crocetin dialdehyde, which is then converted by an unknown aldehyde dehydrogenase to crocetin. A proteome from saffron stigma was released recently and, based on the expression pattern and correlation analyses, five aldehyde dehydrogenases (ALDHs) were suggested as possible candidates to generate crocetin from crocetin dialdehydes. We selected four of the suggested ALDHs and analyzed their expression in different tissues, determined their activity over crocetin dialdehyde, and performed structure modeling and docking calculation to find their specificity. All the ALDHs were able to convert crocetin dialdehyde to crocetin, but two of them were stigma tissue-specific. Structure modeling and docking analyses revealed that, in all cases, there was a high coverage of residues in the models. All of them showed a very close conformation, indicated by the low root-mean-square deviation (RMSD) values of backbone atoms, which indicate a high similarity among them. However, low affinity between the enzymes and the crocetin dialdehyde were observed. Phylogenetic analysis and binding affinities calculations, including some ALDHs from Gardenia jasmonoides, Crocus sieberi, and Buddleja species that accumulate crocetin and Bixa orellana synthetizing the apocarotenoid bixin selected on their expression pattern matching with the accumulation of either crocins or bixin, pointed out that family 2 C4 members might be involved in the conversion of crocetin dialdehyde to crocetin with high specificity. [ABSTRACT FROM AUTHOR]

• In this study, we analyzed RNAseq data from different saffron cultivars to explore its virome. • We found 17 broadly distributed viruses, some not previously described. • This analysis provides a tool for future studies for saffron phytosanitary improvement. Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 52 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered 7 novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae , and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity. [ABSTRACT FROM AUTHOR]