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

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

Metabolite and target transcript analyses during Crocussativus stigma development

Saffron, the desiccated stigmas of Crocussativus, is highly appreciated for its peculiar colour, flavour and aroma. Several studies have been conducted with the spice, but little is known about the evolution of volatile and non-volatile compounds generated during the development of the stigma. In this study, we have followed these compounds, with special attention to those of isoprenoid origin (carotenoids and monoterpenes), which are responsible for the organoleptic properties of saffron. The main compounds that accumulated throughout stigma development in C.sativus were crocetin, its glucoside derivatives and picrocrocin, all of which increased as stigmas reached a fully developed stage. The volatile composition of C.sativus stigmas changed notably as stigmas developed with each developmental stage being characterized by a different volatile combination. In red stigmas, β-cyclocitral, the 7,8 cleavage product of β-carotene, was highly produced, suggesting the implication of both β-carotene and zeaxanthin in crocetin formation. As stigmas matured, hydroxy-β-ionone and β-ionone were produced while safranal, the most typical aroma compound of the processed spice, was only detected at low levels. However, a safranal-related compound 2,2,2-trimethyl-2-cyclohexene-1,4-dione (4-oxoisophorone) increased rapidly at the anthesis stage and also in senescent stigmas. Monoterpenes were mainly emitted at the time of anthesis and the emission patterns followed the expression patterns of two putative terpene synthases CsTS1 and CsTS2. Fatty acid derivates, which predominated at the earlier developmental stages, were observed at low levels in later stages.     

A review of the chemistry and uses of crocins and crocetin,the carotenoid natural dyes in saffron,with particular emphasis on applications as colorants including their use as biological stains

The perennial flowering plant, saffron crocus (Crocus sativus L.), is the source of the most expensive spice in the world. The dried stigmas of saffron flowers are the source of a natural dye, saffron, which has been used from ancient times for dyeing silk and fabric rugs, and for painting; it also has been used for cooking and in medicine. The yellow compounds present in the dye include crocins, which are 20-carbon water soluble glycosyl derivatives of the carotenoid, crocetin, and the dicarboxylic acid itself. We review the chemistry of these compounds and discuss various applications of saffron as a natural dye. We review in particular the use of saffron or its constituents in histopathologic techniques.     

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, β-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.

A carotenoid cleavage dioxygenase from Bixa orellana elicits crocin production in Solanaceae.     

Ultrastructure of chromoplasts and other plastids in Crocus sativus L. (Iridaceae)

Saffron (Crocus sativus L. Iridaceae) chromoplasts and other plastids were studied by electron microscope to determine their structure, origin and pigment localization. Plastids from pistils of floral buds and flowers at anthesis, dried and decoloured stigmas, and green and senescent leaves were examined. Results indicated that mature saffron chromoplasts occur in the red parts of stigmas and have a reticulo-tubular structure. They contain a reticulum of tubules and plastoglobules. Tubules formed dilated vesicles mainly while plastoglobules appeared numerous and scattered on the whole chromoplast. Chromoplasts appeared in red stigma of very young floral buds. They originated from amyloplasts, the only plastids occurring in the colourless basal portion of style, as well as in the parenchyma of ovary and corm. Transition forms of plastid as amylo-chromoplast, occur in the yellow parts of stigma and style. Senescent leaves did not show plastids with structure similar to the chromoplast of red stigma. Red pigmented and scented stigmas might cooperate in saffron reproduction by attracting pollinator.     

Interaction of tRNA with Safranal,Crocetin, and Dimethylcrocetin

Abstract

Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional therapeutic. The biological activity of saffron in modern medicine is in development. Its numerous applications as an anti-oxidant and anti-cancer agent are due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). The aim of this study was to examine the interaction of transfer RNA with safranal, crocetin, and dimethylcrocetin in aqueous solution at physiological conditions. Constant tRNA concentration (6.25 mM) and various drug/tRNA (phosphate) molar ratios of 1/48 to 1/8 were used. FT-IR and UV-Visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants and the effects of drug complexation on the stability and conformation of tRNA duplex. External binding mode was observed for safranal crocetin and dimethylcrocetin, with overall binding constants K_safranal = 6.8 (± 0.34) × 10³ M^?1, K_CRT = 1.4 (± 0.31) × 10⁴ M^?1, and K_DMCRT = 3.4 (± 0.30) × 10⁴ M^?1. Transfer RNA remains in the A-family structure, upon safranal, crocetin and dimethylcrocetin complexation.     

Developmental and stress regulation of gene expression for a 9-cis-epoxycarotenoid dioxygenase, CstNCED, isolated from Crocus sativus stigmas

Oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. ABA has been associated with dormancy and flower senescence, while also regulating plant adaptive responses to various environmental stresses. An NCED gene, CstNCED, was cloned from Crocus sativus stigmas. The deduced amino acid sequence of the CstNCED protein shared high identity with other monocot NCEDs, and was closely related to the liliopsida enzymes. At the N-terminus of CstNCED a chloroplast transit peptide sequence is located. However, its expression in chloroplast-free tissues suggested localization in other plastid types. The relationship between expression of CstNCED and the endogenous ABA level was investigated in the stigma and corms, where it was developmentally regulated. The senescence of the unpollinated stigma is preceded by an increase in ABA levels and CstNCED expression. In corms, a correlation was observed between CstNCED expression and dormancy. Furthermore, CstNCED expression was correlated with the presence of zeaxanthin in the dormant corms. When detached C. sativus leaves and stigmas were water and salt stressed, increases in CstNCED mRNA were observed. The results provided evidence of the involvement of CstNCED in the regulation of ABA-associated processes such as flower senescence and corm dormancy in monocotyledonous saffron.     

Distribution of Assimilates from Various Source Leaves During the Development of Gladiolus grandiflorus

The distribution pattern of the products of photosynthesis wasstudied in gladiolus plants (Gladiolus grandiflorus cv Eurovision)in four stages of development I, plants having a very younginflorescence still enclosed between the leaves; II, plantswith a young inflorescence just emerged from the leaves, III,plants at full bloom, IV, plants with young fruits. The first,third or sixth foliage leaf was labelled with ¹⁴CO₂, and subsequentdistribution in the plant was determined Results were expressedas a percentage of translocated ¹⁴C accumulated by each partof the plant which gives a measure of its ‘sink strength’,or as ‘relative sink activity’ (RSA) which is independentof the size of the indicated organ. There are two competing sinks in the developing gladiolus—theinflorescence and the new corm. When RSA is the criterton theinflorescence constitutes the main sink irrespective of thesource leaf from the first stage until flowering. With the subsequentwilting of the flowers and fruit set RSA of the inflorescencedeclines rapidly and the new corm becomes the main sink When‘sink strength’ is the criterton it appears thatthe inflorescence acts as a very weak sink when it is youngand becomes increasingly stronger until flowering and then declinessteeply. Sink strength of the corm declines during the developmentof the inflorescence and then increases again steeply with wiltingof the flowers and fruit set. There are small differences betweenthe various source leaves. The young new corm acts as a strongsink for the lower foliage leaf and progressively weaker forupper leaves. Gladiolus grandiflorus, flower development, corm, assimilates distribution, sink strength, relative sink activity     

INDIRECT SELECTION OF STIGMA POSITION IN IPOMOPSIS AGGREGATA VIA A GENETICALLY CORRELATED TRAIT

Experimental manipulation of a trait can be used to distinguish direct selection from selection of correlated traits and to identify mechanisms of selection. Here we use experiments to investigate phenotypic selection of stigma position in angiosperm flowers. In natural populations of the subalpine herb Ipomopsis aggregata, plants with more strongly exserted stigmas receive more pollen per flower, indicating selection favoring stigma exsertion during the pollination stage of the life cycle. We pose four hypotheses for this association, two involving direct selection on stigma position and two involving indirect selection of a correlated floral trait. The first three hypotheses were tested using hand pollinations that mimicked natural hummingbird visitation, and by presenting captive hummingbirds with a series of flowers that differed in stigma and anther positions, sex ratio, and presence of anthers. In these experiments, pollen deposition either was independent of stigma exsertion or was highest on inserted stigmas, suggesting direct selection against exserted stigmas. In natural populations, however, stigma exsertion is highly correlated with time spent by the protandrous flowers in the pistillate phase. When we manipulated the latter trait in the field, pollen deposition increased with duration of exposure to hummingbirds, indicating indirect selection for stigma exsertion. Stigma exsertion and time spent in the pistillate phase are genetically and phenotypically correlated, as shown by a quantitative genetic experiment conducted in the field with paternal half sibships. Our results suggest that the evolution of stigma position can be driven by selection of a genetically correlated trait.     

Pollination and stigma wounding: same response, different signal?

In Petunia hybrida flowers, both pollination and stigma woundinginduced a transient Increase in ethylene production and hastenedcorolla senescence. Ethylene production by different flowerparts was measured in situ using laser photoacoustic (LPA) spectroscopy.In pollinated flowers, ethylene was exclusively produced bythe stigma/style region whereas wounding of the stigma Inducedethylene production both by the stigma/style region and by theremaining flower parts. In aminoethoxyvinylglycine (AVG)-treatedflowers, subsequent treatment of the unwounded stigma with 1-aminocyclopropane-1-carboxylicacid (ACC) induced ethylene production exclusively by the stigma/styleregion whereas treatment of a previously wounded stigma withACC induced a simultaneous increase in ethylene production bythe stigma/style region and the remaining flower parts. Theseresults suggest that following stigma wounding, either ACC orethylene is involved in inter-organ communication. Followingpollination, the signal is apparently not directly related toethylene. In vivo ACC oxidase activity of most flower parts, includingthe gynoecium, was higher in light than in dark. Light or darkdid not influence the relative contributions of stigma/styleand remaining flower parts to the total pollination, woundingor ACC-induced ethylene production, indicating that ACC is nottranslocated. Both in excised styles and intact flowers, radiolabelledACC and its analogue -aminoisobutyric acid (AIB), applied eitherto an intact or wounded stigma, were largely immobile confirmingthat ACC is not likely to play a role in inter-organ signalling. The results collectively suggest that following stigma wounding,translocation of ethylene may be the signal responsible forinitiation of corolla senescence; following pollination thesignal is not directly related to ethylene. Key words: 1-Aminocyclopropane-1-carboxylic acid (ACC), ethylene, flower senescence, Petunia hybrida, pollination, stigma wounding     

Nutrient resorption of two evergreen shrubs in response to long-term fertilization in a bog

Plant resorption of multiple nutrients during leaf senescence has been established but stoichiometric changes among N, P and K during resorption and after fertilization are poorly understood. We anticipated that increased N supply would lead to further P limitation or co-limitation with N or K [i.e. P-(co)limitation], decrease N resorption and increase P and K resorption, while P and K addition would decrease P and K resorption and increase N resorption. Furthermore, Ca would accumulate while Mg would be resorbed during leaf senescence, irrespective of fertilization. We investigated the effect of N, P and K addition on resorption in two evergreen shrubs (Chamaedaphne calyculata and Rhododendron groenlandicum) in a long-term fertilization experiment at Mer Bleue bog, Ontario, Canada. In general, N addition caused further P-(co)limitation, increased P and K resorption efficiency but did not affect N resorption. P and K addition did not shift the system to N limitation and affect K resorption, but reduced P resorption proficiency. C. calyculata resorbed both Ca and Mg while R. groenlandicum resorbed neither. C. calyculata showed a higher resorption than R. groenlandicum, suggesting it is better adapted to nutrient deficiency than R. groenlandicum. Resorption during leaf senescence decreased N:P, N:K and K:P ratios. The limited response of N and K and the response of P resorption to fertilization reflect the stoichiometric coupling of nutrient cycling, which varies among the two shrub species; changes in species composition may affect nutrient cycling in bogs.     

Influence of frost on nutrient resorption during leaf senescence in a mangrove at its latitudinal limit of distribution

The latitudinal distribution of mangrove species is limited mainly by low temperature. Leaf scorch and massive leaf fall are the predominant symptoms of frost damage. Nutrient resorption during leaf senescence is an important adaptation mechanism of mangroves. Abnormal defoliation disturbs nutrient resorption. We evaluated the effects of frost on nutrient loss of mangroves and the protective effects of warmer seawater inundation on reducing nutrient loss. On January 14, 2009, the most cold-tolerant mangrove Kandelia obovata at its naturally latitudinal limit (Fuding, China, 27°17??N) was exposed to freezing temperature (?2.4°C) for 4 h (minimum ?2.8°C). The freezing air temperature occurred during flood tide, resulting that the flooded shoots were protected by warmer seawater. Frost caused 31.3% and 13.0% defoliation on the exposed shoots and the flooded shoots, respectively. Frost restricted nutrient resorption during leaf senescence. K. obovata resorbed 61% N and 42% P during normal leaf senescence, respectively. However, frost-damaged leaves only resorbed 13% N and 10% P during the course, respectively. Foliar N:P molar ratios were <31, suggesting N limitation. Tidal inundation can partially protect mangroves from frost damage. Reduced nutrient resorption efficiency and massive leaf fall caused by frost add pressure to mangroves under nutrient limitation at their latitudinal limits.     

不同光周期对西红花开花和花丝品质的效应比较

为筛选促进西红花开花和提高花丝品质的最佳光周期,以3种不同规格(20～25 g、25～30 g、30～35 g)西红花种球为材料,设置4种光周期处理(8 h/16 h、10 h/14 h、12 h/12 h、14 h/10 h),考察光周期对西红花种球鲜重变化、主芽生长、展叶数量、开花后营养物质含量以及花朵性状和花丝品...     

Changes in ethylene production and 1-aminocyclopropane-1-carboxylic acid content of pollinated carnation flowers

Pollination of flowers of standard carnation (Dianthus caryophyllus L. cv. White Sim) with pollen from flowers of miniature carnations (D. caryophyllus L. cv. Exquisite) caused them to wilt irreversibly within 1 to 2 days. Pollination stimulated a sequential increase in ethylene production by stigmas, ovaries, receptacles, and petals of the flowers. The ACC content of the stigmas increased rapidly in the first few hours after pollination. The possibility that subsequent production of ethylene by other parts of the flower is stimulated by translocated ACC is discussed. Ethylene production and ACC content of other parts of the flower reached their maximum 24 h after pollination. The petal tissues contributed the bulk of the ethylene productionper flower thereafter. There appears to be a qualitative difference between the enzyme in the stigmas converting ACC to ethylene and that in other parts of the flower.     

The pollination biology and breeding system of Monarda fistulosa (Labiatae)

Summary Successful cross-pollination of Monarda fistulosa is the result of a complex interaction among flower opening, the pollen-bearing areas of the pollinators and/or their behavior, and the maturation of the stigmas. The flowers open continuously from 0800–2000 h providing a temporally predictable rich source of nectar and pollen. Recently opened flowers may reduce the ability of bees to discriminate between resource rich and poor patches and encourage systematic foraging within patches. The continuous opening of flowers coupled with protandry also results in some flowers of most capitula being in the staminate and others in the pistillate phase. Autogamy is highly unlikely due to strong protandry and the spatial separation of anthers and stigmas. Geitonogamy, at least that mediated by Bombus is unlikely because the pollen is spread over a relatively large area of the wings, which reduces the likelihood of a stigma contacting just deposited pollen. Because pollen is transferred from the much smaller coxal area of Anthophora and other bees that mistake the stigmas of early pistillate phase flowers for stamens some geitonogamy seems inevitable. However, the delayed receptivity of young stigmas to self-pollen decreases the likelihood of self-pollen germinating on such stigmas. Older stigmas are equally receptive to self- and cross-pollen and the number of pollen grains germinating and pollen tubes reaching the base of the style increases with flower age.