Saffron - American Chemical Society

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Saffron: Its Phytochemistry, Developmental Processes, and Biotechnological Prospects Oussama Ahrazem,†,‡ Angela Rubio-Moraga,† Sergio G. Nebauer,§ Rosa Victoria Molina,§ and Lourdes Gómez-Gómez*,† †

Instituto Botánico, Departamento de Ciencia y Tecnologı ́a Agroforestal y Genética, Facultad de Farmacia, Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain ‡ Fundación Parque Cientı ́fico y Tecnológico de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain § Departamento de Biologı ́a Vegetal, Universidad Politécnica de Valencia, 46071 Valencia, Spain ABSTRACT: The present state of knowledge concerning developmental processes and the secondary metabolism of saffron, Crocus sativus L. (Iridaceae), along with the genes involved in these processes so far known, is reviewed. Flowers and corms constitute the most valuable parts of saffron. Corm and flower development are two key aspects to be studied in saffron to increase the yield and quality of the spice, to raise its reproductive rate, and to implement new production systems. Important knowledge about the physiology of flowering and vegetative growth has been acquired in recent years, but there is still only limited information on molecular mechanisms controlling these processes. Although some genes involved in flower formation and meristem transition in other species have been isolated in saffron, the role of these genes in this species awaits further progress. Also, genes related with the synthesis pathway of abscisic acid and strigolactones, growth regulators related with bud endodormancy and apical dominance (paradormancy), have been isolated. However, the in-depth understanding of these processes as well as of corm development is far from being achieved. By contrast, saffron phytochemicals have been widely studied. The different flower tissues and the corm have been proved to be an important source of phytochemicals with pharmacological properties. The biotechnological prospects for saffron are here reviewed on the basis of the discovery of the enzymes involved in key aspects of saffron secondary metabolism, and we also analyze the possibility of transferring current knowledge about flowering and vegetative propagation in model species to the Crocus genus. KEYWORDS: apocarotenoids, corm development, flowering, flavonoids, saffron, saponins, stigmas, genes

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INTRODUCTION The genus Crocus comprises around 85−100 species, primarily distributed in the MediterraneanEurope and western Asia. Turkey and the Balkan peninsula accumulate the major number of species and subspecies. Greece alone contributes to ca. 40% of the world’s wild Crocus diversity,1 whereas a total of 32 species (18 of them being endemic) are included in Turkey flora.2 Several countries also have representatives of some Crocus species: Italy (10 species), Spain (6 species), Hungary (6 species), among others. The genus Crocus is divided into two subgenera:3 the subgenus Crocus, which includes all species except one, and C. banaticus, which is the unique member of the subgenus Crociris. The subgenus Crocus is further divided into two sections: section Crocus and section Nudiscapus. This subdivision of the genus is based on morphological and cytological characters,3 as well as genetic analysis.4 Crocus sativus belongs to the series Crocus, commonly known as saffron. Within this series the plants have finely fibrous corm tunics, autumnal flowers, white or unmarked transparent membranous flaccid bracts usually not closely sheathing the perianth-tube, yellow anthers, and three style branches, which are usually red due to the accumulation of crocins5 and often expended at the apex, entire or at most fimbriate.3 C. sativus L. (Figure 1), a triploid geophyte, is a sterile plant propagated by corms and adapted to overcome the dry dormant period of summer in the form of an underground corm.3 © XXXX American Chemical Society

Figure 1. Most valuable parts of Crocus sativus: (A) saffron flowers; (B) corms; (C) stigma.

Historical data suggest that saffron is a very ancient cultivation dating back from 2500 to 1500 BCE.6 The place of origin, although not clear, is probably Greece, Iran, or Asia Minor, later Received: June 30, 2015 Revised: September 25, 2015 Accepted: September 28, 2015

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DOI: 10.1021/acs.jafc.5b03194 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry spreading to India, China, the Mediterranean basin, and eastern Europe.7 Saffron is widely used mainly as a spice and as a coloring and flavoring agent in both the agro-food and cosmetic industries. The saffron spice is a complex mixture of volatile and nonvolatile compounds and carotenoid derivatives, which all contribute to the overall aroma and flavor of the spice.8,9 In addition to its color, taste, and aroma, saffron displays a variety of health benefits.10 Saffron shows analgesic and sedative properties11 and is a potential anticancer agent.12,13 Furthermore, saffron is beneficial for the treatment of mental disorders such as depression14 and dementia.15

Every sprouting bud in the mother corm has the potential to produce a new corm, but saffron corms show only a few sprouting apical buds plus many axillary dormant buds. Furthermore, there is a heterogeneity in bud emergence and development, which results in heterogeneous flower emergence.17 This process must therefore be controlled to establish industrial production. In this context, environmental and genetic factors affecting corm development, dormancy, and sprouting, as well as the identification of environmental signals and the genetic and regulatory mechanisms that promote or inhibit flowering in saffron, constitute the most relevant processes to be elucidated for implementing a technological breakthrough and achieving agronomical improvements in this crop.

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AGRONOMICAL IMPROVEMENT OF SAFFRON PRODUCTION The ideal condition for improving crop yield would be the optimization of all metabolic cues together with the environmental conditions where the crop develops.16 For such a purpose, the rates of all important processes should be optimized and also their interactions and duration, which are generally determined by genetic mechanisms often influenced by the environment. Cultivation of saffron occurs in temperate regions. The life cycle of saffron is adapted to the climate of the Mediterranean regions and is quite similar in all producing countries, but with wide differences in the timing of events.17 In the Mediterranean region, saffron flowers in autumn, after the formation of replacement corms at the base of the shoots is initiated. At the beginning of the dry season (April−May), the leaves senesce and wither, and the corms go into dormancy. The transition from the vegetative to the reproductive stage occurs before summer in the apex of the buds of underground corms, which do not have roots or foliage leaves.17 Not all of the newly developed corms are able to produce flowers. Flower formation is directly related to corm size.18 Therefore, the adequate production of corms is extremely important to guarantee flower production. During the past decades, the area traditionally dedicated to saffron cultivation in many European countries has fallen dramatically,19,20 mainly due to the intensive manual labor required for saffron harvest and processing (0.25−2.5 million flowers ha−1). However, in recent years this tendency has reversed, and saffron production in Europe and other developed countries outside Eurasia, for example, in Tasmania (Australia), is increasing, with a specific emphasis on the production of highquality saffron. A technological breakthrough to increase the quality of saffron cultivation would entail the cultivation under controlled conditions in containers to facilitate the mechanization of flower harvesting and stigma separation.21 To achieve this, the flowering season must be extended as much as possible to maximize the use of harvesting installations and thus reduce overall installation and running costs. Thus, an in-depth knowledge about the regulation of the flowering process could help to increase the yield of the spice. Another important factor that limits the areas in which saffron is cultivated is the difficulty in obtaining high-quality propagation material with guaranteed levels of purity, homogeneity, and health. It is a slow-growing species from which three to four daughter corms per mother corm are obtained each season under natural field conditions. Low multiplication and fungal diseases of corms reduce the productivity and quality, thereby restraining the availability of planting material and the widespread use of improved genotypes obtained in the future.

WHAT IS KNOWN ABOUT FLOWERING AND CORM DEVELOPMENT IN SAFFRON? Saffron Flowering. The transition from the vegetative to the reproductive stage in saffron occurs early in July when the apex becomes dome shaped and increases in size. This process is followed by the formation of the leaf primordia. By mid July the sheathing leaves begin to grow relatively quickly. Then follows the bract primordia stage, the formation of stamen primordia, the initiation of the perianth, and the formation of gynoecium. All of the flower parts are already differentiated by the end of August (Figure 2). High temperatures are required to release bud

Figure 2. Saffron flower development during summer: (A) resting bud at the end of June; (B) stamen formation in early-mid August; (C) first whorl of tepals is already initiated in the flower at mid-late August; (D) gynoecium has reached half of the length of the stamens in mid September. Scale bars in panels A−C = 0.5 mm. Scale bar in panel D = 1 mm.

dormancy and for flower initiation, which is optimal between 23 and 27 °C21 and occurs during early spring to mid summer, depending on location.22−24 The release of bud dormancy could be accelerated by curing the corms at 30 °C for a short time. Storage of corms at 2 °C after flower initiation results in a timedependent abortion of those flowers already initiated. Corms stored in the cold before flower initiation will form flowers when incubated after storage at 21−25 °C. When corms are maintained for a long time at very low temperatures (1−10 °C), the meristem does not give rise to flower primordia but to a new B


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Figure 3. Development of saffron daughter corms (DC) in the field (Valencia, Spain): relationship between mother corm (MC) and daughter corm sizes (flowering took place in November): (A) in early December; (B) by the end of January; (C) in mid February; (D) by the end of February; (E) by the end of April.

corm.19 Root development and flower emergence occur in late autumn at a markedly lower temperature, in the range of 15−17 °C.21,25 This contrasts with spring-flowering bulbous species, which require a warm−cold−warm temperature sequence to flower.26 The research carried out so far on saffron has produced extensive knowledge related to the influence of temperature on flower induction and flowering and the extension of the harvesting period over several months,17,21,22 but this strategy has reached its peak. Few works27,28 have addressed the role of hormones on flowering. In these works flowering development is related to GA activity. The involvement of other environmental or endogenous factors has not been studied, and the molecular mechanisms controlling these processes are largely unknown. The CEN/ TFL1-like gene (CENTRORADIALIS/TERMINAL FLOWER1) has been cloned and characterized in saffron.29 CsatCEN/TFL1 transcripts were detected in corms and flower organs but not in leaves, and their role in saffron flowering control is still unclear. Three FLOWERING LOCUS T (FT)-like genes, designated CsatFT1-like, CsatFT2-like, and CsatFT3-like, have been isolated.30 The expression analysis indicated differences in the expression of the three FT-like genes in different organs. However, the expression has been measured in flowers already developed and in immature flowers, but not in the run-up to floral transition. Hence, the role of the FT-like genes awaits further progress. In relation to the molecular mechanisms controlling flower development in cultivated Crocus, different full-length cDNA sequences encoding MADS-box transcription factor proteins involved in flower formation have been cloned and characterized. Homologues of the major ABC and E types from Crocus have been isolated and named Csat AP1/FUL,31 CsatAP2,29 CsatAP3 and CsatPI,32 and CsatAG33 and CsatSEP3.34 However, the expression analysis did not always show consistent results with the ABC model, and a functional understanding has to be clarified. For example, the three isolated CsatAP1 genes were expressed in leaves, as well as in the three mature flower parts: tepals, stamen, and carpel. Furthermore, an expanded expression

of class B genes in whorls 1 and 4 was also detected. The expression of class B genes into whorl 1 fits the modified ABC model for monocots; however, the additional expression is not consistent with this model. Corm Development. There is limited knowledge about the physiology of vegetative development of saffron (Figure 3). The relative contribution of different carbon sources (leaf photosynthetic activity and mother corm reserves) to the growth of vegetative organs and the influence of internal and external factors on the photosynthetic processes sustaining corm growth have been studied.19 After flowering, most of the remaining reserves in the mother corm are depleted during root and leaf development. Once these organs reach their maximum size, the growth of replacement corms is initiated, and this development primarily relies on photosynthesis. During this vegetative development, the reserves from the mother corm contribute to only 10% of the biomass. The photosynthetic rate is consistently high and constant (26 μmol m−2 s−1) throughout the year, as has also been reported in C. vernus.35,36 Plants with larger corms show reduced photosynthetic rate, although a significantly higher total leaf area is present.19 A sink capacity limitation arises during growth of replacement corms because the increase of biomass is limited before total leaf senescence. A reduction in the demand of photoassimilates can lead to sugar accumulation in leaves, which in turn can promote leaf senescence.37 The studies carried out in C. vernus, a spring ephemeral, support the hypothesis that corm sink strength is the most important factor determining leaf duration.35,38 Differences in sink strength have been associated with differential carbon partitioning between sugar forms in the newly formed corms. A higher availability in hexoses at the beginning of the development of the daughter corms would affect cell division and elongation and, thus, higher corm size that allowed prolonged sink activity.36 However, an in-depth knowledge about the environmental and genetic factors controlling the final size and the sink strength of the saffron corm still seems far from being achieved. Sprouting Control in Saffron. Sprouting is another important aspect in saffron research. As stated previously, C


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Journal of Agricultural and Food Chemistry every sprouted bud produces a corm and factors affecting apical dominance (para-dormancy) are highly important for corm production. However, the control of dormancy (endodormancy) is also crucial in solving the many problems associated with storage and distribution of corms and for extending the flowering and harvesting periods. Physiological and molecular studies support the concept that the mechanisms of flowering induction and dormancy are interrelated.39 It has been suggested that dormancy-associated MADS-box (DAM) genes induced by cold temperatures in leafy spurge negatively regulates FT or similar genes, which regulate growth cessation and dormancy induction. Most of these studies were performed on species showing winter dormancy, where dormancy was regulated by low temperature and a short photoperiod. However, saffron shows summer dormancy, which is common in typical bulbous species. This plant strategy has been correlated with superior survival and high persistence under severe drought conditions. Summer dormancy is therefore of great interest because intense drought and heat are increasing due to climate change and may affect plant persistence. However, this type of dormancy is relatively uncharacterized at a molecular level. Recently, the characterization of summer dormancy in the bulbous species Narcissus tazetta and the role of FTs in summer dormancy has been reported.40 In saffron, FT genes and CEN/ TFL1-like genes that could be related with the dormancy pathway have been isolated.30 Dormancy interacts at some level with the mechanisms involved in cell cycle regulation, which also involves hormonal signals.39,41 Studies of the hormonal regulation of dormancy in geophytes are abundant.42 Clear roles for abscisic acid (ABA), such as inducing dormancy, have been revealed. ABA has been recognized to control dormancy in the corm tissue of C. sativus, where it declines at the time of dormancy release.28 It has been proposed that ABA is synthesized from carotenoids (C40) in plants, and three genes that participate in ABA biosynthesis have been isolated and characterized in several plant species. They encode zeaxanthin epoxidase (ZEP43), 9-cis-epoxycarotenoid dioxygenase (NCED44), and abscisic aldehyde oxidase (AAO45). ZEP catalyzes the epoxidation of zeaxanthin to produce epoxycarotenoid; NCED catalyzes the cleavage reaction of epoxycarotenoids to produce xanthoxin (the first C15 intermediate) and is considered to be the regulatory enzyme of ABA biosynthesis; and AAO catalyzes the final step of ABA biosynthesis, which converts ABA aldehyde to ABA. Homologue genes for ZEP, AAO, and NCED have been identified in saffron. However, only one NCED gene, CsNCED, has been studied in detail.46 CsNCED is expressed in floral and corm tissues. In stigmas, CsNCED transcript abundance closely mirrors ABA content with a maxima a day after anthesis, and it has been suggested that the ABA levels in the stigma could be related to the initiation of the senescent process in this tissue.47 During corm development, zeaxanthin levels are correlated with the increase in CsNCED expression, suggesting a coordinate regulation of the carotenoid and ABA biosynthetic pathways during corm dormancy.46 Mature saffron corms usually show one to three apical dominant buds, which will sprout in the following season plus many axillary dormant buds.22 Each axillary bud has the same developmental potential as the primary shoot apical meristem in its ability to produce a growing shoot axis (Figure 4). However, axillary buds enter a dormant state after forming only a few leaves. Plant hormones are major players in the control of axillary bud outgrowth. In particular, auxin, strigolactone, and cytokinin

Figure 4. Apical dominance in saffron: (A) corm with the main bud sprouting and dormant axillary buds; (B) axillary bud growth after removal of the main bud.

are involved in this control.48 In saffron, auxin is supplied from the apical bud and, together with the strigolactones (SL), represses axillary bud outgrowth. By contrast, the cytokinins, produced in the adventitious roots, directly induce the sprout of the axillary buds.49 Gibberellins (GAs) seem to be involved in apical sprout growth after dormancy break, but not in dormancy maintenance or release.28 SLs are derived from the oxidative cleavage of carotenoids and have been implicated in many processes including root growth, root hair and stem elongation, lateral root formation, adventitious rooting, secondary growth, leaf expansion, leaf senescence, and drought and salinity responses but most prominently in shoot branching.48 SLs seem to act systemically to dampen the polar auxin transport stream, which enhances competition among buds for a common auxin sink.50 To date, four loci involved in SL biosynthesis and three loci involved in SL signaling have been identified.51−53 The first steps of SL biosynthesis involve isomerization by the plastidial-isomerase, followed by the sequential cleavage activity of CCD7 and CCD8 carotenoid cleavage dioxygenases. MAX1 encodes a cytP450 predicted to act downstream from CCD7 and CCD8 and is required for synthesis of active SL. Among all of the identified components of the strigolactone biosynthetic pathway, CsCCD7 and CsCCD8 genes have been recently isolated and studied in saffron.54 SL production related to bud dormancy in saffron seems to be controlled at the CsCCD8 level. In addition, CsCCD8 seems to play an important role in the development of the vasculature of the axillary buds in saffron. Recent evidence shows that SLs positively regulate cambial activity, and this function is conserved among species. Saffron is a monocot without cambial activity. However, given the relationship between cambial and procambial cells, it can be postulated that SLs could also positively regulate procambium activity and the development of vascular tissues in growing buds of monocots.

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THE PHYTOCHEMISTRY OF SAFFRON: COMPOUNDS IDENTIFIED FROM SAFFRON FLOWERS AND CORMS Apocarotenoids in Saffron Flowers, Biosynthesis, and Properties. Apocarotenoids are the products of the oxidative cleavage of carotenoids by specific carotenoid cleavage oxygenases (CCDs) that recognize and specifically cleave one or two double bonds.55 If sufficient numbers of conjugated double bonds are maintained in the cleavage products they can show coloration, like their parent carotenoids. This is the case of the crocetin molecule (8,8′-diapo-8,8′-carotene-dioic acid) (Figure 5), widely used as a colorant in foods and cosmetics. The crocetin D


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Figure 5. Pathway for crocin and picrocrocin biosynthesis in saffron stigmas.

with 200 fewer amino acids. For CsZCD a 7−8/7′−8′ cleavage activity in vitro was reported.70 However, CsZCD lacks important residues and domains for a dioxygenase activity,74 and no activity was found for this CsZCD in later trials.68,75 Mining of an EST database from C. sativus stigmas has provided further information on potential saffron biosynthetic genes but no candidate for a novel CCD with 7−8/7′−8′ cleavage specificity.76 Therefore, the previously isolated enzymes were analyzed against a battery of different carotenoids, including zeaxanthin and lutein,77 and an enzyme, CsCCD2, with a confirmed specificity for the 7−8/7′−8′ double bonds was identified, showing 97% identity with the initially identified CsCCD1b68 (Figure 6A). On the basis of the in vitro results, lutein and zeaxanthin, but not β-carotene, are good substrates for CsCCD2, and the availability or accessibility to the enzyme would determine the specificity. Accumulation of apocarotenoids in saffron is stigma specific and appears to be developmentally regulated.73 Higher levels of saffron apocarotenoids increase as stigmas develop and reach their peak in the red stage,73 and it is likely that the genes involved in their biosynthesis may be stimulated during the process and enhanced in the stigma tissue. CsCCD1b and CsCCD2 expressions are limited to the stigma tissue and detected in the earlier developmental stages, when crocetin accumulation takes place.68 Analysis of saffron EST collections obtained from the stigma at different developmental stages revealed that CsCCD2 ESTs were more highly represented in libraries obtained from early stages.77 Recently, homologues of CsCCD2 have been identified in spring Crocus species, which accumulate crocins in stigmas and tepals.78 All together, the CCD2 enzymes constitute a new CCD subfamily with no homologues identified in other plant species so far. Apocarotenoid Properties. The stigmas of C. sativus have been used in folk medicine to alleviate different health

molecule is further modified by the activity of glucosyltransferases,56,57 which add different numbers of glucose molecules to produce characteristic crocins, these being the major components of the stigmas of saffron and which confer solubility.58,59 Crocins are widely present in the species of the genus Crocus,60 but have been also identified in Buddleja officinalis,61 in Jacquinia angustifolia,62 in Coleus forskolii,63 in Nyctanthes abor-tristis,64 in the fruits of Artocarpus heterophyllus,65 Gardenia jasminoides,66 and even in the cyanobacterium Microcystis.67 Crocetin is formed from the enzymatic cleavage of zeaxanthin on the 7−8/7′−8′ double bonds (Figure 6).68,69 The resulting products are crocetindial and hydroxyl-β-cyclocitral, which is further glucosylated to form picrocrocin (β-D-glucopyranosidehydroxyl-β-cyclocitral), the degradation product of which is the odor-active safranal.69 Carotenoid biosynthesis, cleavage activities, and the expression of corresponding genes have been studied during the development of the stigma of C. sativus. A total of five CCD genes belonging to two classes of cleavage enzymes were identified from C. sativus.68,70,71 Heterologous expression of CsCCD1a, CsCCD1b, CsCCD4a, CsCCD4b, and CsCCD4c sequences reduced accumulation of β-carotene in Escherichia coli strains engineered for the accumulation of this carotenoid.68,72 As for volatile cleavage products produced in these strains, only C13 β-ionone could be detected, but no C10 apocarotenoids, whereas a 9−10/9′−10′ cleavage activity for both types of enzymes is predicted (Figure 6). Analysis of stigma volatiles in different stages of flower development indicated high production of β-ionone shortly before and at anthesis.68,73 In the plant, CsCCD1a and CsCCD4a/b differ if their subcellular location is cytosolic or plastidial, respectively.68 CsCCD4a sequences, most likely constituting an allelic variant of CsCCD4b (98% of identity) turned out to be identical to a Crocus ZCD sequence,70 albeit CsZCD is a truncated sequence compared to CsCCD4a, E


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Figure 6. Carotenoid cleavage dioxygenases identified in the stigma of saffron: (A) phylogenetic relationships among the isolated CsCCDs; (B) cleavage activity for the isolated CsCCDs enzymes.

problems.79 Recent pharmacological studies have demonstrated that crude extracts from saffron and purified chemicals possess antitumor effects, display anti-inflammatory properties, and counteract atherosclerosis and hepatic damage.11,12 In studies on animal models, it has been shown that crocetin has several

pharmacological properties, including antioxidant,80,81 antiinflammatory,82 antiatherosclerotic,83 insulin resistance improvement,84 attenuation of physical fatigue,85 and sleep.86 Crocetin also alters the growth of cancer cells by inhibiting replication, inducing apoptosis, and enhancing the antioxidative system.87 F


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3,7-O-diglucoside. These flavonols are present in the flowers of Crocus sp. from the series Crocus, but absent in Crocus species from other series, and could be used for quimiotaxonomic purposes. Flavonoid Properties. In plants, flavonoids have many diverse functions including UV protection, defense, allelopathy, flower coloring to attract pollinators, auxin transport inhibition, plant−microorganism communication, and regulation of reactive oxygen species, and in many species they are required for pollen viability.105 A number of biological processes, such as signal transduction, transcriptional regulation, and cell-to-cell communication, are also influenced by flavonoids. In addition, they play crucial roles in human nutrition, and many therapeutic benefits of flavonoids are known in animal systems. Flavonoids have antioxidant, anti-inflammatory, antitumor, antiproliferative, cardioprotective, and pro-apoptotic activities.106−109 The main flavonoid present in saffron tepals is kaempferol-3O-β-D-glucopyranosyl-(1−2)-β-D-glucopyranoside (kaempferol 3-O-β-sophoroside).103 Interestingly, this glucosylated flavonoid is not present in all Crocus species, and its presence is relegated to a few series, including the Crocus series. Kaempferol 3-O-βsophoroside has been reported to have analgesic activity.110 Furthermore, kaempferol 3-O-β-sophoroside inhibits the vascular endothelial inflammatory responses and is considered a promising target for the treatment of vascular diseases such as atherosclerosis, shock, heart attack, and sepsis.111,112 The identification in saffron of the first UGT involved in the formation of this glucosylated flavonoid opens new possibilities for the production of this compound in other crop species or in other heterologous systems.

Crocetin shows neuroprotective effects.88,89 Crocetin inhibits in vitro amyloid-β aggregation,90 and a clinical pilot study revealed significant improvements in cognition after the treatment of mild-to-moderate Alzheimer’s disease patients with crocetin.91 Crocins had protective effects on neuronal injury,92 and attenuated the symptoms of obsessive compulsive disorder, a common psychiatric disorder defined by the presence of obsessive thoughts and repetitive compulsive actions.93 Safranal, the main constituent of the volatile oil fraction, attenuated cerebral ischemia94 and retinal degeneration.95,96 Flavonoids in Saffron Flowers. Flavonoids are among the best-characterized plant secondary metabolites in terms of chemistry, coloration mechanism, biochemistry, genetics, and molecular biology. With a basic structure of C6−C3−C6, they are widely distributed among land plants. Flavonoids are the result of the contribution of two pathways, the flavonoid branch of the phenylpropanoid and the acetate−malonate metabolic pathway. Although the central pathway for flavonoid biosynthesis is conserved in plants, depending on the species, a group of enzymes, such as isomerases, reductases, hydroxylases, and several Fe2+/2-oxoglutarate-dependent dioxygenases, modify the basic flavonoid skeleton, leading to the different flavonoid subclasses.97 Finally, transferases modify the flavonoid backbone with sugars, methyl groups, and/or acyl moieties, modulating the physiological activity of the resulting flavonoid by altering its solubility, reactivity, and interaction with cellular targets.98 In C. sativus, flavonols are usually glycosylated at their 3-OH, 7-OH and 4′-OH positions,8,99,100 producing a complex pattern of flavonols. In C. sativus stigmas three main glucosides of kaempferol have been identified: kaempferol 7-O-sophoroside, kaempferol 3-O-sophoroside-7-O-glucopyranoside, and kaempferol 3,7,4′-triglucoside. By contrast, 21 different glycosides of isorhamnetin, kaempferol, myricetin, naringenin, quercetin, tamarixetin, and taxifolin have been identified in tepals,101 kaempferol and kaempferol glycosides being the most dominant class of flavonoids in this tissue (70 and 90% of the total content of flavonoids) followed by quercetin glycosides (5−10%).102 In pollen kaempferide, a methylated kaempferol and the isorhamnetin glycosides isorhamnetin-3,4′-diglucoside, isorhamnetin-3-O-robinobioside, and isorhamnetin-3-β-D-glucoside are detected.100 In addition, the anthocyanins delphinidin, petunidin, and malvidin have been identified in tepals, with different sugar substitutions,102 and are responsible for their blue color. Transcriptome analysis of saffron stigmas has allowed the identification of genes encoding for key enzymes of the flavonol pathway, and putative UDP-glycosyltransferase enzymes have been identified (data not shown). To date three genes encoding flavonol glycosyltransferases have been characterized in saffron.99,103 The saffron CsGT45 (UGT75P1) protein showed specificity toward flavonoid aglycones and was found to be active on the C-7 position of kaempferol, kaempferol 7-O-sophoroside being the most abundant kaempferol glucoside present in the stigma tissue. The UGT707B1 enzyme was recently characterized as a flavonol-3-O-glucoside: 2″-glucosyltransferase. The expression of UGT707B1 in the floral organs of C. sativus allowed the accumulation of a specific set of flavonols that influence the transport of the plant hormone auxin and could be responsible for the characteristic morphology of the stigma.103 The last UGT characterized in saffron involved in flavonoid glucosylation is UGT703B1.104 This enzyme recognizes isorhamnetin and kaempferol as substrates in vitro. In addition, UGT703B1 expression was found to be highly correlated with the presence of kaempferol 7-O-biglucoside-3-O-β-glucoside and isorhamnetin-

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SAPONINS IN SAFFRON CORMS Saponin Biosynthesis. Saponins are a widespread group of terpenoids. Saponins are important plant defense compounds, which are determinant for disease resistance due to their antimicrobial, fungicidal, allelopathic, and insecticidal activities.113 Humans exploit saponins for their medicinal properties and antimicrobial activity. The composition of saponins depends on the genetic background of the plant material, the tissue type, and the age and physiological state of the plant as well as environmental factors.114 The name saponin is derived from sapo, soap in Latin, because the surfactant properties produce soap-like foams in aqueous solution. This trait is the result of the amphiphilic nature of saponins due to linkage of the lipophilic sapogenin to hydrophilic saccharide side chains. According to the chemical nature of the aglycone (known as sapogenin), the saponins are divided into steroidal and triterpenoid saponins. Steroidal saponins are produced especially in some families of the monocotyledons, for example, Agavaceae, Alliaceae, Asparagaceae, Convallariaceae, Dioscoreaceae, Liliaceae, Smilacaceae, and Trilliacea. In dicotyledons, steroidal saponins are detected only in a few families, for example, Fabaceae, Solanaceae, and Scrophulariaceae.115 Cholesterol is considered the starting molecule for the biosynthesis of steroidal saponins.116 The first committed step in the biosynthesis of triterpenoid saponins and phytosterols is the cyclization of 2,3oxidosqualene by oxidosqualene cyclases (OSGs). The high number of possibilities for establishing different internal linkages during cyclization gives rise to a vast array of different triterpene skeletons.117 Following formation of basal sapogenin backbone structures mediated by OSCs, various modifications take place prior to glycosylation, the most common being the addition of small functional groups such as aldehyde, hydroxyl, keto, and G


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Figure 7. Structures of the triterpenic saponins, azafrines 1 and 2, identified in the corm.

advantages in terms of preservation of the genetic characteristics of the plant, but makes any genetic improvement difficult. Variability studies with saffron from different countries and locations showed the absence of or very low variability among entries.124−126 This insufficient genetic variability makes the use of this plant difficult for selection programs,127,128 whereas the induction of genetic variability through mutagenesis has been addressed using corms at different developmental stages. Even though mutations have been identified as resulting from experimental as well as natural mutagenesis,129,130 these are not maintained because they are not heritable. In C. sativus, many experiments have been performed to obtain seeds131 and in vitro regenerated plants132 without success. As an alternative, researchers have been trying to elucidate how the triploid species was generated to identify the progenitors or at least the closest relatives of C. sativus. An autotriploid origin was initially suggested for saffron. However, an allotriploid origin from two wild species has been proposed in different papers,102,133,134 C. cartwrightianus being one of the accepted progenitors.5,34,135 Nevertheless, no such effort has been undertaken to exploit the closet relatives and hybrid generation. A first EST collection from saffron was developed in 200776 comprising 6603 high-quality ESTs from a saffron mature stigma cDNA. The rapid evolution of next-generation DNA sequencing technologies has allowed the analysis of the transcriptome of six different developmental stages from saffron stigma (unpublished results). This will allow deciphering key players involved in one of the most important processes in saffron from the biotechnological point of view such as apocarotenoid metabolism. However, it is necessary to point out that one of the most important problems in applying modern biotechnological approaches to saffron and geophytes in general is the lack of high-frequency transformation systems. Further efforts should be made to work in that direction.

carboxyl moieties at different positions of the backbone, most of them introduced by cytochromes P450 enzymes.118 Saponin glycosylation involves sequential activity of different UGTs.98 Typical triterpenoid saponin glycosylation patterns consist of oligomeric sugar chains, two to five monosaccharide units, which are most often linked at position C3 and/or C28. In saffron, two triterpenic saponins have been identified in the corm, azafrines 1 and 2119 (Figure 7). Azafrine 1 contains 3-O-βD-glucopyranosiduronic acid echinocystic acid as prosapogenin, and azafrine 2 contains 3-O-β-D-galactopyranosiduronic acid echinocystic acid as prosapogenin. Both prosapogenins are substituted at the C28 position with a pentasaccharide consisting of β-D-galactopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→2)[β-D-xylopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)-β-Dfucopyranose, and a 4-O-di-α-L-rhamnopyranosyl-3,16-dihydroxy-10-oxo-hexadecanoyl group is linked to C-4 of the β-Dfucopyranosyl residue. Azafrines 1 and 2 are localized in the external part of the corms, suggesting their involvement in plant defense.119,120 Saponin Properties. Saponins or saponin-containing plant extracts are exploited by the pharmaceutical industry. Several saponins, including the ones identified in saffron, exhibit pharmacological activities and thus attract attention as a target for drug discovery. Saponins are valuable adjuvants, and the first saponin-based vaccines have been introduced commercially.121 In addition, a recent study demonstrates the potential use of azafrines 1 and 2 in human immunotherapeutic vaccines,122 and they can also be used for further industrial applications, for instance, preservatives, flavor modifiers, and agents for removal of cholesterol from dairy products123

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PROSPECTS FOR IMPROVING AND INNOVATING SAFFRON PRODUCTION: THE ROLE OF GENOMIC TOOLS AND BIOTECHNOLOGICAL APPLICATIONS All over the world saffron is known as one cultivar. The continuous vegetative cultivation of saffron offers certain H


Review

Journal of Agricultural and Food Chemistry Secondary Metabolism and Biotechnological Applications. The current state of the art regarding secondary metabolism in saffron has been discussed above. The identification of the key enzymes involved in apocarotenoid formation represents a very important tool for future biotechnological applications in other crop species where the substrates for crocetin formation are present. This is the case of maize and tomato, where introduction of these enzymes will provide intense red colors and higher nutritional quality, due to the higher bioavailability of the saffron apocarotenoids compared with carotenoids.136 In addition, rice and tobacco are also good candidates to increase the production of crocins, providing added value to the crop, in the case of rice, or using the plant as a cheaper way to produce crocetin in sufficient quantities for different industrial applications. In this regard, it is also important to consider other Crocus species as alternative sources of saffron apocarotenoids, paying special attention to those that accumulate crocins in both stigma and tepals,60 providing an attractive crop from an economic standpoint, due to the reduction of processing costs. With regard to corm saponins, the genetic machinery required for the elaboration of this important family of plant secondary metabolites is as yet largely uncharacterized in saffron, despite the considerable commercial interest in this important group of natural products. The development of transcriptomes from the corm combined with metabolomic analyses will allow the identification of key enzymes involved in their biosynthesis. Nevertheless, still little is known about the genetics that control quantitatively and qualitatively the biosynthesis of these secondary metabolites in saffron and its allies. In addition, knowledge of the regulation of these pathways is practically nonexistent, and such knowledge is of crucial importance to bypass the possibilities of low product yield of these secondary metabolites in other plants or plant cell cultures. Control of Flower Development. Understanding flowering regulation at the molecular level in saffron, and also in other spring and autumn Crocus, would provide the potential for more accurately controlling both flowering time and the development of transcriptional markers for flower initiation to increase genetic variation of saffron for flowering time from spring Crocus using biotechnological and omics tools. Although several key flowering genes (FT, CEN/TFL1, and AP1) have been found in saffron, understanding floral inductive pathways in Crocus requires not only the identification of some possible regulator genes but a precise knowledge of their expression in the different developmental stages and their control by environmental or internal factors. To date, we are far from achieving this knowledge. Limited information is available on the flower transition of bulbous species, which require high temperature for flower differentiation in species such as saffron. The results obtained by Li et al.137 working on Narcissus tazetta, a bulbous species that requires high temperature for flower differentiation, showed that Narcissus FLOWERING LOCUS T1 (NFT1) transcripts were abundant during flower initiation in mature bulbs and were up-regulated by high temperature, suggesting that NFT1 possibly takes part in flower transition control in response to this factor. Given the similarities in the floral induction process of saffron and narcissus, the same upregulation of FT by high temperature could be expected to happen in saffron, although this needs to be confirmed. Also, the role of CsatCEN/TFL1 needs to be clarified. Although the involvement of TFL1 genes in floral transition

and axillary meristem identity has been demonstrated in other monocot species,138 their role in saffron is not clear. As previously mentioned, treating dry saffron corms with GA in June−July accelerated flower formation and promoted the formation of additional flower buds from undifferentiated meristems. Furthermore, some observations point out that GAs and nutrients act through a common signaling pathway to promote flowering.139 This pathway controlling flowering in response to gibberellin and nutrients is highly recommended to be studied in saffron. Because flower differentiation takes place in summer when the corm is located underground with no foliage or roots, the involvement of the photoperiod and vernalization pathways seems unlikely, but they should not be entirely ruled out. Another important aspect is related to the decision to bloom, which requires lower temperatures than for flower induction. The genetic control of this process has not yet been studied. Understanding flower induction in saffron could reveal ways to extend the harvesting period, but the introduction of genetic variation from other Crocus species by means of biotechnological tools should also be considered. Furthermore, the development of new genotypes from C. cartwrightianus should not be excluded. There is a high variability for flowering time in the Crocus genus. This fact enables us to establish a research line aimed at acquiring in-depth knowledge about the differences in the flowering processes at the molecular level between autumn and spring Crocus species and making use of this knowledge to extend the saffron flowering period. Another interesting biotechnological prospect has been pointed out by Tsaftaris and co-workers.33 It would be desirable to have mutants without stamen formation in saffron flowers, because removal of stamens and separation of stigmas by hand is very labor intensive, leading to high costs. More interesting would be the transformation of stamens to carpel, which should increase saffron production in a single flower. However, it is not at all clear that saffron production would be doubled. The identification of saffron flower development genes that has been already carried out, as mentioned above, will facilitate the achievement of these objectives, and the usefulness of these materials can be tested. Control of Saffron Sprouting. The understanding and control of dormancy are critical for effectively managing crop production, commercial handling, and storage. Control of endodormancy in saffron store corms is a requirement for staggering the production, and reliable markers for endo-dormancy in saffron and other geophytes are one of the most important issues in horticultural practice. The saffron genes ZEP, AAO, NCED, and dormancy-associated MADS-box (DAM) genes could be used as markers to determine the exact time at which environmental or internal factors trigger dormancy. It is evident that the application of molecular techniques is indispensable for clarifying the precise mechanism(s) of dormancy induction and release in bulbous species, and the establishment of such a model system is crucial for further research on dormancy of bulbous species.42 Another important aspect of dormancy regulation in the Crocus genus is the differential response of various plant organs to environmental signals as has been pointed out by Okubo.42 The genus includes both synanthous and hysteranthous species. In both types, flower initiation occurs in summer, and the initiation orders of the leaves and flowers are similar. However, a time interval takes place between leaf elongation and anthesis in hysteranthous species, when compared to synanthous species (Figure 8). Hence, different organs might vary in their dormancy I


Review


selective forces that act on this trait and extend our understanding of the regulation of photosynthesis, ultimately increasing the productivity of crops. Several experimental manipulations suggest that photosynthesis and sink utilization of carbohydrates are closely coordinated.146 The regulation of carbon partitioning between sources and sinks has a great impact on plant growth and yield and is directly linked to the mechanisms of assimilate transport and allocation. Depending on the main sugar transported and the phloem loading/unloading mechanisms working, different transporters can be involved (e.g., SWEET, H+/sucrose symporters). The biotechnological manipulation of the transport systems has been reported to positively alter carbon and also nitrogen partitioning and consequently improve crop yields, although pleiotropic effects limiting these gains can also occur.147 However, at this time no information is available about sugar transport in Crocus. The growth capacity of the sinks is a measure of its strength and depends on sink size and activity. Sink size includes cell number and cell size and is potentially regulated by hormones (gibberellins and cytokinins) along with the supply of assimilates. Bigger corms are formed when photoassimilate availability is higher.19 Despite the genetic basis, corm size has been proposed to be influenced in Crocus by the content and type of soluble sugar content at earlier stages of corm development.36 Sink activity includes multiple components and key enzymes of carbohydrate metabolism and storage, thus maintaining an assimilate gradient and transport between source and sink.148 Sucrose is the main transported sugar in many species, and several key enzymes are involved in carbon partitioning. Cell wall and vacuole invertases (INV) in apoplastic unloaders have been related to sink strength, although other activities such as sucrose phosphate phosphatase (SPP), sucrose synthase (SS), or sucrose phosphate synthase (SPS) may also be involved. In addition, final storage products, for example, starch, are the ultimate sinks, and genes involved in their metabolism also influence sink strength.149 The integration of carbon assimilation and growth of organs is highly regulated, and hormonal regulation of source-sink relations has been well stated, although many aspects are not fully understood.148,150 Another interesting approach would be the use of transcription factors to alter the activity of several genes involved in a given process. Several studies in recent years point out this possibility to enhance photosynthesis and biomass production.150

Figure 8. Crocus genus includes both (A) synanthous (e.g., Crocus sativus) and (B) hysteranthous (e.g., Crocus nudif lorus) species.

requirements, and the process might be controlled by both external and internal signals. The absence of leaves at flowering time is of great interest in saffron collection to make harvest work easier, and the understanding of that differential response of leaves and flowers in hysteranthous species takes on great importance. The rate of propagation in saffron is directly related to axillary meristem formation and outgrowth, and it has been shown that there is a control exerted by the main shoot apex of the corm over the outgrowth of the lateral buds mediated by a hormonal balance. Auxin and strigolactones inhibit bud outgrowth, and cytokinins promote it. A bud-specific gene that promotes bud arrest could be the key element to integrate the bud outgrowth pathway.140 Such a gene exists in maize (Teosinte branched 1TB1),141 and there is evidence that the gene BRANCHED1 (BRC1) acts as an integrator of branching signals within axillary buds of Arabidopsis.142 BCR1 is up-regulated by SLs and downregulated by cytokinins. Because outgrowth of axillary buds seems to be the major limiting factor in vegetative propagation of bulbs and corms, the SL signaling pathway and BRC1-like genes are important targets of choice to study and optimize vegetative propagation.140 Improvement of Corm Growth. The manipulation of assimilate partitioning and allocation within the plant is a key factor in the improvement of corm growth. This goal could be achieved through biotechnological approaches based on the identification of the involved genes and regulatory pathways. Photosynthesis efficiency can be improved through different targets. In relation to light reactions, it could be achieved by increasing the efficiency of light capture, energy transduction, and photoprotective processes, and for carbon fixation reactions by reducing limitations in the diffusion of CO2 to the site of carboxylation (e.g., mesophyll conductance), in the capacity for carboxylation/oxygenation of Rubisco, and in the regeneration of RuBP.143 In addition, other processes involving carbon metabolism, such as starch and sucrose synthesis, photorespiration, or respiration, also determine net photoassimilate production.144 Despite the potential of biotechnological approaches in optimizing the photosynthetic process, the study of natural genetic variation on photosynthesis within species and between related species is a valuable undertaking.145 A survey performed on 60 different accessions of C. sativus in the field revealed significant differences in the photosynthetic rate and in the quantum efficiency of photosystem II (unpublished data). Research is currently being conducted to characterize the sources of variation within the photosynthetic process and to assess the relationship to biomass production in saffron and related Crocus species. This would provide insight into the

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CONCLUSIONS On the basis of this review, we conclude that the molecular and physiological approaches to analyzing and addressing saffron production processes and the exploitation of their secondary metabolism products remain largely unexplored. It is essential to integrate crop physiology and genomic approaches to provide a useful framework in which synergies can be explored between technology and systems-oriented approaches. This review provides a starting point for key aspects in saffron biotechnology.

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AUTHOR INFORMATION

Corresponding Author

*(L.G.-G.) E-mail: [email protected]. Phone: +34 967599200. J


Review

Journal of Agricultural and Food Chemistry Funding

(21) Molina, R. V.; Valero, M.; Navarro, Y.; Garcia-Luis, A.; Guardiola, J. L. Low temperature storage of corms extends the flowering season of saffron (Crocus sativus L.). J. Hortic. Sci. Biotechnol. 2005, 80, 319−326. (22) Molina, R.; Valero, M.; Navarro, Y.; Garcia-Luis, A.; Guardiola, J. L. The effect of time of corm lifting and duration of incubation at inductive temperature on flowering in the saffron plant (Crocus sativus L.). Sci. Hortic. 2004, 103, 79−91. (23) Milyaeva, E. L.; Azizbekova, N. S. Cytophysiological changes in course of development of stem apices of saffron crocus. Soviet Plant Physiol. 1978, 25, 227−233. (24) Koul, K. K.; Farooq, S. Growth and differentiation in the shoot apical meristem of saffron plant (Crocus sativus L.). J. Indian Bot. Soc. 1982, 63, 153−160. (25) Plessner, O.; Negbi, M.; Ziv, M.; Basber, D. Effects of temperature on the flowering of the saffron crocus (Crocus sativus L.): induction of hysteranthy. Isr. J. Bot. 1989, 38, 7. (26) Wilkins, H. F. Crocus vernus, Crocus sativus. In Handbook of Flowering; Halevy, A. H., Ed.; CRC Press: Boca Raton, FL, USA, 1985; Vol. 2, pp 350−355. (27) Azizbekova, N. S. H.; Milyaeva, E. L.; Lobova, N. V.; Chailakhyan, M. K. H. Effects of gibberellin and kinetin on formation of flower organs in saffron crocus. Fiziol. Rast 1978, 25, 6. (28) Farooq, S.; Koul, K. K. Changes in gibberellin-like activity in corms of saffron plant (Crocus sativus L.) during dormancy and sprouting. Biochem. Physiol. Pflanz. 1983, 178, 685−689. (29) Tsaftaris, A.; Pasentsis, K.; Kalivas, A.; Michailidou, S.; Madesis, P.; Argiriou, A. Isolation of a CENTRORADIALIS/TERMINAL FLOWER1 homolog in saffron (Crocus sativus L.): characterization and expression analysis. Mol. Biol. Rep. 2012, 39, 7899−7910. (30) Tsaftaris, A.; Pasentsis, K.; Argiriou, A. Cloning and characterization of FLOWERING LOCUS T-like genes from the perennial geophyte saffron crocus (Crocus sativus). Plant Mol. Biol. Rep. 2013, 31, 1558−1568. (31) Tsaftaris, A.; Pasentsis, K.; Iliopoulos, I.; Polidoros, A. N. Isolation of three homologous AP1-like MADS-box genes in crocus (Crocus sativus L.) and characterization of their expression. Plant Sci. 2004, 166, 1235−1243. (32) Tsaftaris, A. S.; Polidoros, A. N.; Pasentsis, K.; Kalivas, A. Cloning, structural characterization, and phylogenetic analysis of flower MADSbox genes from crocus (Crocus sativus L.). Sci. World J. 2007, 7, 1047− 1062. (33) Tsaftaris, A. S.; Pasentsis, K.; Polidoros, A. N. Isolation of a differentially spliced C-type flower specific AG-like MADS-box gene from crocus (Crocus sativus) and characterization of its expression. Biol. Plant. 2005, 49, 499−504. (34) Tsaftaris, A.; Pasentsis, K.; Makris, A.; Darzentas, N.; Polidoros, A.; Kalivas, A.; Argiriou, A. The study of the E-class SEPALLATA3-like MADS-box genes in wild-type and mutant flowers of cultivated saffron crocus (Crocus sativus L.) and its putative progenitors. J. Plant Physiol. 2011, 168, 1675−1684. (35) Badri, M. A.; Minchin, P. E. H.; Lapointe, L. Effects of temperature on the growth of spring ephemerals: Crocus vernus (L.) Hill. Physiol. Plant. 2007, 130, 67−76. (36) Lundmark, M.; Vaughan, H.; Lapointe, L. Low temperature maximizes growth of Crocus vernus (L.) Hill via changes in carbon partitioning and corm development. J. Exp. Bot. 2009, 60, 2203−2213. (37) Wingler, A.; Masclaux-Daubresse, C.; Fischer, A. M. Sugars, senescence, and ageing in plants and heterotrophic organisms. J. Exp. Bot. 2009, 60, 1063−1066. (38) Lapointe, L. How phenology influences physiology in deciduous forest spring ephemerals. Physiol. Plant. 2001, 113, 151−157. (39) Horvath, D. Common mechanisms regulate flowering and dormancy. Plant Sci. 2009, 177, 523−531. (40) Feng, Y.; Zhu, L.; Pan, T.; Guo, Z.; Zhong, X.; Ding, A.; Pan, D. Characterization of summer dormancy in Narcissus tazetta var. chinensis and the role of NtFTs in summer dormancy and flower differentiation. Sci. Hortic. 2015, 183, 109−117.

This work was supported by the Spanish Ministerio de Economı ́a y Competitividad (BIO2013-44239-R). Notes

The authors declare no competing financial interest.

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