Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma

Gómez-Gómez, Lourdes; Parra-Vega, Verónica; Rivas-Sendra, Alba; Seguí-Simarro, José M.; Molina, Rosa Victoria; Pallotti, Claudia; Rubio-Moraga, Ángela; Diretto, Gianfranco; Prieto, Alicia; Ahrazem, Oussama

doi:10.3390/ijms18010076

Cited by 48 publications

(34 citation statements)

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“…Coexpression and correlation network were performed by calculating pairwise Pearson correlations between each UGT gene and picrocrocin; subsequently, Fisher's Z ‐transformation was used to determine the statistical significance of the pairwise correlations. Finally, significant correlations, and with P > |0.60|, at least, were used to draw a correlation network, by using Cytoscape 3.7.0 software with a force‐directed layout, and as previously described (Gomez‐Gomez et al ., ; Ahrazem et al ., ).…”

Section: Methodsmentioning

confidence: 99%

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus)

Diretto¹,

Ahrazem

Rubio-Moraga

et al. 2019

New Phytologist

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Summary Saffron, a spice derived from the dried red stigmas of Crocus sativus, is one of the oldest natural food additives. The flowers have long red stigmas, which store significant quantities of the glycosylated apocarotenoids crocins and picrocrocin. The apocarotenoid biosynthetic pathway in saffron starts with the oxidative cleavage of zeaxanthin, from which crocins and picrocrocin are derived. In the processed stigmas, picrocrocin is converted to safranal, giving saffron its typical aroma. By a targeted search for differentially expressed uridine diphosphate glycosyltransferases (UGTs) in Crocus transcriptomes, a novel apocarotenoid glucosyltransferase (UGT709G1) from saffron was identified. Biochemical analyses revealed that UGT709G1 showed a high catalytic efficiency toward 2,6,6‐trimethyl‐4‐hydroxy‐1‐carboxaldehyde‐1‐cyclohexene (HTCC), making it suited for the biosynthesis of picrocrocin, the precursor of safranal. The role of UGT709G1 in picrocrocin/safranal biosynthesis was supported by the absence or presence of gene expression in a screening for HTCC and picrocrocin production in different Crocus species and by a combined transient expression assay with CsCCD2L in Nicotiana benthamiana leaves. The identification of UGT709G1 completes one of the most highly valued specialized metabolic biosynthetic pathways in plants and provides novel perspectives on the industrial production of picrocrocin to be used as a flavor additive or as a pharmacological constituent.

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Section: Methodsmentioning

confidence: 99%

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus)

Diretto¹,

Ahrazem

Rubio-Moraga

et al. 2019

New Phytologist

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“…The function of Or has been associated to the differentiation of proplastids and/or other noncolored plastids into chromoplasts, providing a metabolic sink for carotenoid www.nature.com/scientificreports www.nature.com/scientificreports/ accumulation 7 . During the development of the stigma in saffron, the increase in size is concomitant with the transformation of amyloplasts into chromoplasts 18 . During the transition from the red to the pre-anthesis stage the amyloplast became undetectable 18 , and the number of plastoglobules and a membranous network increased 34 , matching the fully developed chromoplasts.…”

Section: Discussionmentioning

confidence: 99%

“…The biosynthesis and accumulation of crocins take place in the stigma and is developmentally regulated. Young undeveloped stigma in saffron and in other Crocus species is white, containing amyloplasts 18,19 . The transition from amyloplast to chromoplast marks the initiation of crocins biosynthesis and their accumulation in the stigma 18 with the presence of a distinctive red color.…”

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confidence: 99%

“…Young undeveloped stigma in saffron and in other Crocus species is white, containing amyloplasts 18,19 . The transition from amyloplast to chromoplast marks the initiation of crocins biosynthesis and their accumulation in the stigma 18 with the presence of a distinctive red color. Crocins biosynthesis in saffron involves a chromoplast-specific carotenoid biosynthetic pathway [20][21][22] , where CsPSY2 is the enzyme catalyzing the first committed step in the carotenoid biosynthesis for the production of the colored apocarotenoids that accumulate in the stigma of saffron, together with CsLCY-B2, CsBCH2 and CCD2 enzymes 20,21,23 .…”

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confidence: 99%

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Differential interaction of Or proteins with the PSY enzymes in saffron

Ahrazem

López

Argandoña

et al. 2020

Sci Rep

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Colored apocarotenoids accumulate at high concentrations in few plant species, where display a role in attraction of pollinators and seed dispersers. Among these apocarotenoids, crocins accumulate at high concentrations in the stigma of saffron and are responsible for the organoleptic and medicinal properties of this spice. Phytoene synthase and Orange protein are key for carotenoid biosynthesis and accumulation. We previously isolated four phytoene synthase genes from saffron with differential roles in carotenoid and apocarotenoid biosynthesis. However, the implications of Orange genes in the regulation of apocarotenoid accumulation are unknown. Here, we have identified two Orange genes from saffron, with different expression patterns. CsOr-a was mainly expressed in vegetative tissues and was induced by light and repressed by heat stress. Both CsOr-a and CsOr-b were expressed in stigmas but showed a different profile during the development of this tissue. The interactions of CsOr-a and CsOr-b were tested with all the four phytoene synthase proteins from saffron and with CsCCD2. None interactions were detected with CCD2 neither with the phytoene synthase 2, involved in apocarotenoid biosynthesis in saffron. The obtained results provide evidence of different mechanisms regulating the phytoene synthase enzymes in saffron by Orange for carotenoid and apocarotenoid accumulation in saffron.Nucleic acid purification and cDNA isolation. Total RNA was isolated from red stigmas 48 , following the manufacturer's protocols (Qiagen, Hilden, Germany). First-strand cDNAs were synthesized by RT from 2 µg of total RNA using an 18-base pair oligo dT primer and a first-strand cDNA synthesis kit (GE Healthcare Life Sciences, www.gelifesciences.com) according to manufacturer's instructions. These cDNAs were used as templates for PCR using specific primers for CsOr-a and CsOr-b (Suppl . Table 1). Thermal cycling parameters were 2 min at 95 °C, 35 cycles of 20 s at 95 °C, 20 s at 60 °C and 1 min 30 s at 72 °C, followed by a final extension of 5 min at 72 °C. The PCR products were separated in a 1% agarose gel, purified, ligated into pGEM-T (Promega, www. promega.com), and then introduced into E. coli. Scientific RepoRtS |(2020) 10:552 | https://doi.

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“…Cleavage of the zeaxanthin molecule at the 7,8 and 7′,8′ double bonds render one molecule of crocetin dialdehyde and two molecules of 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) (Ahrazem et al, 2017, 2016c; Frusciante et al, 2014). Crocetin dialdehyde is further transformed to crocetin by the action of aldehyde dehydrogenase (ALDH) enzymes (Demurtas et al, 2018; Gómez-Gómez et al, 2018, 2017). Next, crocetin serves as substrate of glucosyltransferase (UGT) activities that catalyze the production of crocins through transfer of glucose to both ends of the molecule (Côté et al, 2001; Demurtas et al, 2018; Moraga et al, 2004; Nagatoshi et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Efficient production of saffron crocins and picrocrocin inNicotiana benthamianausing a virus-driven system

Martí¹,

Diretto²,

Aragonés³

et al. 2019

Preprint

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22Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, 23 for the color and the unique taste of the saffron spice, known as red gold due to its 24 high price. Several studies have also shown the health-promoting properties of these 25 compounds. However, their high costs hamper the wide use of these metabolites in 26 the pharmaceutical sector. We have developed a virus-driven system to produce 27 remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana 28 plants in only two weeks. The system consists of viral clones derived from tobacco 29 etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes 30 from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues 31 demonstrated that the sole virus-driven expression of C. sativus CsCCD2L or B. 32 davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. 33 2 Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of 34 crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. 35 In an attempt to improve apocarotenoid content in N. benthamiana, co-expression 36 of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene 37 synthase (PaCrtB) and saffron -carotene hydroxylase 2 (BCH2), was performed 38 using the same viral system. This combinatorial approach led to an additional crocin 39 increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. 40 Considering that saffron apocarotenoids are costly harvested from flower stigma 41 once a year, and that Buddleja spp. flowers accumulate lower amounts, this system 42 may be an attractive alternative for the sustainable production of these appreciated 43 metabolites. 44 45

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Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma

Cited by 48 publications

References 49 publications

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus)

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus)

Differential interaction of Or proteins with the PSY enzymes in saffron

Efficient production of saffron crocins and picrocrocin inNicotiana benthamianausing a virus-driven system

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