A New Glycosyltransferase Enzyme from Family 91, UGT91P3, Is Responsible for the Final Glucosylation Step of Crocins in Saffron (Crocus sativus L.).

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.

Transcriptomic analysis of saffron at different flowering stages using RNA sequencing uncovers cytochrome P450 genes involved in crocin biosynthesis.

Saffron is a well-known Chinese traditional herb, and crocin biosynthesis is related to the yield and quality of saffron. This study aimed to screen differentially expressed genes (DEGs) in saffron at different flowering stages and identify cytochrome P450 (CYP) genes involved in crocin biosynthesis. Saffron samples at different flowering stages were used for RNA sequencing, and DEGs between the samples at three days before the flowering stage (- 3da) and two days after the flowering stage (+ 2da) were screened. Thereafter, significantly differentially expressed CYP genes were identified, and CYP gene expression at different flowering stages and in various tissues of saffron was determined using real-time quantitative polymerase chain reaction (RT-qPCR). After sequencing and analysis, 1508 DEGs between the samples at - 3da and + 2da were identified, including 487 upregulated and 1021 downregulated genes, which were enriched in 16 biological processes, 5 cellular components, 3 molecular functions, and 11 KEGG pathways, including protein processing in endoplasmic reticulum, pentose and glucuronate interconversions, starch and sucrose metabolism, estrogen signaling pathway, and mitogen-activated protein kinase signaling pathway. In addition, 12 significantly differentially expressed CYP genes were identified. The RT-qPCR results showed that CYP76C4, CYP72A15, CYP72A219, CYP97B2, CYP714C2, CYP71A1, CYP94C1, and CYP86A8 were all expressed in the pistils, and CYP72A219, CYP72A15, CYP97B2, CYP71A1, and CYP86A8 were highly expressed in the pistils. Our study established a transcriptome library of saffron and found that CYP72A219, CYP72A15, CYP97B2, CYP71A1, and CYP86A8 may be candidates involved crocin biosynthesis in saffron.

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 ß-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.

Construction of Escherichia coli cell factories for crocin biosynthesis.

BACKGROUND: Crocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, ß-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. RESULTS: In this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC-ESI-MS. CONCLUSIONS: A heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.

Transcriptome analysis reveals novel enzymes for apo-carotenoid biosynthesis in saffron and allows construction of a pathway for crocetin synthesis in yeast.

Crocus sativus is generally considered the source of saffron spice which is rich in apo-carotenoid compounds such as crocins, crocetin, picrocrocin, and safranal, which possess effective pharmacological activities. However, little is known about the exact genes involved in apo-carotenoid biosynthesis in saffron and the potential mechanism of specific accumulation in the stigma. In this study, we integrated stigmas at different developmental stages to perform in-depth transcriptome and dynamic metabolomic analyses to discover the potential key catalytic steps involved in apo-carotenoid biosynthesis in saffron. A total of 61 202 unigenes were obtained, and 28 regulators and 32 putative carotenogenic genes were captured after the co-expression network analysis. Moreover, 15 candidate genes were predicted to be closely related to safranal and crocin production, in which one aldehyde dehydrogenase (CsALDH3) was validated to oxidize crocetin dialdehyde into crocetin and a crocetin-producing yeast strain was created. In addition, a new branch pathway that catalyses the conversion of geranyl-geranyl pyrophosphate to copalol and ent-kaurene by the class II diterpene synthase CsCPS1 and three class I diterpene synthases CsEKL1/2/3 were investigated for the first time. Such gene to apo-carotenoid landscapes illuminate the synthetic charactersistics and regulators of apo-carotenoid biosynthesis, laying the foundation for a deep understanding of the biosynthesis mechanism and metabolic engineering of apo-carotenoids in plants or microbes.

Functional characterization of flavonoid 3'-hydroxylase, CsF3'H, from Crocus sativus L: Insights into substrate specificity and role in abiotic stress.

Stress-responsive dihydroxy flavonoids exhibit capability to inhibit the accretion of reactive oxygen species (ROS). The formation of these dihydroxy flavonols is catalyzed by flavonoid hydroxylases which are among the rate limiting enzymes of flavonoid biosynthesis pathway. Although flavonoid hydroxylases have been identified in several plant species but their role in abiotic stress is not explicitly documented. In the present study we report identification of all the flavonoid biosynthesis pathway genes of Crocus sativus and their expression profiling. We also report functional characterization of flavonoid 3' hydroxylase (CsF3'H) and attempt to explore its physiological role in vitro and in planta. The results indicated that CsF3'H is 1608 bp long encoding 535 amino acids. Docking and enzyme kinetic studies revealed that CsF3'H catalyzes hydroxylation of naringenin and dihydrokaempferol to eriodictoyl and dihydroquercetin respectively, but exhibits higher affinity for naringenin. Further, CsF3'H showed comparatively higher expression in floral tissues particularly stigma and its expression was significantly enhanced in response to UV-B, dehydration and salinity stress indicative of its role in stress. The expression of CsF3'H was associated with concomitant accumulation of eriodictoyl and dihydroquercetin. Transient overexpression of CsF3'H in Nicotiana benthamiana leads to the accumulation of substantial amounts of eriodictoyl and dihydroquercetin. Further, it was observed that transient expression of CsF3'H conferred tolerance to UV-B and dehydration stress as was evident from higher chlorophyll and soluble sugar and lower MDA contents. Taken together, these results suggest that CsF3'H confers tolerance to UV-B and dehydration in planta through synthesis of dihydroflavonols.

Expression and Interaction Analysis among Saffron ALDHs and Crocetin Dialdehyde.

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.

Elucidation and functional characterization of CsPSY and CsUGT promoters in Crocus sativus L.

The dried stigmas of Crocus sativus constitute the saffron, which is considered to be the costliest spice of the world. Saffron is valuable for its constituents, which are mainly apocarotenoids. In order to enhance the production of apocarotenoids, it is imperative to understand the regulation of apocarotenoid biosynthetic pathway. In C. sativus, although the pathway has been elucidated, the information regarding the regulation of the pathwaygenes is scanty. During the present investigation, the characterization of promoters regulating the expression of two important genes i.e. CsPSY and CsUGT was performed. We successfully cloned the promoters of both the genes, which were functionally characterized in Crocus sativus and Nicotiana tabaccum. In silico analysis of the promoters demonstrated the presence of several important cis regulatory elements responding tolight, hormonesand interaction with transcription factors (TFs). Further analysis suggested the regulation of CsPSY promoter by Abscisic acid (ABA) and that of CsUGT by Gibberellic acid (GA). In addition, we also observed ABA and GA mediated modulation in the expression of significant TFs and CsPSY and CsUGT transcripts. Overall, the study addresses issues related to regulation of key genes of apocarotenoid pathway in C.sativus.

Multiplex Fluorescent, Activity-Based Protein Profiling Identifies Active α-Glycosidases and Other Hydrolases in Plants.

With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (Arabidopsis thaliana), these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident α-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident α-mannosidase-II Hybrid Glycosylation1 (HGL1), both of which trim N-glycans on glycoproteins. We detected the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of α-glycosidases generates pH-dependent signals that can be suppressed by α-glycosidase inhibitors in a broad range of plant species. To demonstrate its use on a nonmodel plant species, we applied ABPP on saffron crocus (Crocus sativus), a cash crop for the production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 67 active glycosidases in saffron crocus stigma, of which 10 are differentially active. We also uncovered massive changes in hydrolase activities in the corms upon infection with Fusarium oxysporum using multiplex fluorescence labeling in combination with probes for serine hydrolases and cysteine proteases. These experiments demonstrate the ease with which active α-glycosidases and other hydrolases can be analyzed through ABPP in model and nonmodel plants.

Functional Characterization of CsBGlu12, a ß-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols.

Glycosylation and deglycosylation are impressive mechanisms that allow plants to regulate the biological activity of an array of secondary metabolites. Although glycosylation improves solubility and renders the metabolites suitable for transport and sequestration, deglycosylation activates them to carry out biological functions. Herein, we report the functional characterization of CsBGlu12, a ß-glucosidase from Crocus sativus. CsBGlu12 has a characteristic glucoside hydrolase 1 family (α/ß)8 triose-phosphate isomerase (TIM) barrel structure with a highly conserved active site. In vitro enzyme activity revealed that CsBGlu12 catalyzes the hydrolysis of flavonol ß-glucosides and cello-oligosaccharides. Site-directed mutagenesis of any of the two conserved catalytic glutamic acid residues (Glu200 and Glu414) of the active site completely abolishes the ß-glucosidase activity. Transcript analysis revealed that Csbglu12 is highly induced in response to UV-B, dehydration, NaCl, methyl jasmonate, and abscisic acid treatments indicating its possible role in plant stress response. Transient overexpression of CsBGlu12 leads to the accumulation of antioxidant flavonols in Nicotiana benthamiana and confers tolerance to abiotic stresses. Antioxidant assays indicated that accumulation of flavonols alleviated the accretion of reactive oxygen species during abiotic stress conditions. ß-Glucosidases are known to play a role in abiotic stresses, particularly dehydration through abscisic acid; however, their role through accumulation of reactive oxygen species (ROS) scavenging flavonols has not been established. Furthermore, only one ß-glucosidase 12 homolog has been characterized so far. Therefore, this work presents an important report on characterization of CsBGlu12 and its role in abiotic stress through ROS scavenging.

Molecular Modeling and Dynamic Simulation of Arabidopsis Thaliana Carotenoid Cleavage Dioxygenase Gene: A Comparison with Bixa orellana and Crocus Sativus.

Carotenoid cleavage dioxygenase (CCD) gene, ubiquitously found in numerous types of plants, are eminent in synthesizing the various volatile compounds (ß-ionone, C13 -norisoprenoid, geranylacetone) known as apocarotenoids. These apocarotenoids have various biological functions such as volatile signals, allelopathic interaction and plant defense. In Arabidopsis genome sequence, four potential CCD genes have been identified namely CCD1, CCD4, CCD7, and CCD8. These four genes give rise to diverse biological functions with almost similar sequence identity. In this investigation, an in silico analysis was proposed to study CCD proteins in Arabidopsis thaliana, aiming at constructing three-dimensional (3D) structure for CCD1 proteins of Bixa orellana and Crocus sativus to observe the structural difference among AtCCD (A. thaliana CCD) proteins. The quality of modeled structures was evaluated using RAMPAGE, PSVS protein validation server and Q Mean server. Finally, we utilised molecular dynamics simulation to identify the stability of the predicted CCD protein structures. The molecular dynamic simulation also revealed that AtCCD4 protein showed lesser stability when compared to other CCD proteins. Overall results from molecular dynamics analysis predicted that BoCCD1, CsCCD1, and AtCCD1 show similar structural characteristics. J. Cell. Biochem. 118: 2712-2721, 2017. © 2017 Wiley Periodicals, Inc.

Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L.

Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases (CCOs). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80 %, respectively. The expression of BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination.

Overexpression of Crocus carotenoid cleavage dioxygenase, CsCCD4b, in Arabidopsis imparts tolerance to dehydration, salt and oxidative stresses by modulating ROS machinery.

Apocarotenoids modulate vital physiological and developmental processes in plants. These molecules are formed by the cleavage of carotenoids, a reaction catalyzed by a family of enzymes called carotenoid cleavage dioxygenases (CCDs). Apocarotenoids like ß-ionone and ß-cyclocitral have been reported to act as stress signal molecules during high light stress in many plant species. In Crocus sativus, these two apocarotenoids are formed by enzymatic cleavage of ß-carotene at 9, 10 and 7, 8 bonds by CsCCD4 enzymes. In the present study three isoforms of CsCCD4 were subjected to molecular modeling and docking analysis to determine their substrate specificity and all the three isoforms displayed high substrate specificity for ß-carotene. Further, expression of these three CsCCD4 isoforms investigated in response to various stresses revealed that CsCCD4a and CsCCD4b exhibit enhanced expression in response to dehydration, salt and methylviologen, providing a clue towards their role in mediating plant defense response. This was confirmed by overexpressing CsCCD4b in Arabidopsis. The transgenic plants developed longer roots and possessed higher number of lateral roots. Further, overexpression of CsCCD4b imparted enhanced tolerance to salt, dehydration and oxidative stresses as was evidenced by higher survival rate, increased relative root length and biomass in transgenic plants as compared to wild type. Transgenic plants also displayed higher activity and expression of reactive oxygen species (ROS) metabolizing enzymes. This indicates that ß-ionone and ß-cyclocitral which are enzymatic products of CsCCD4b may act as stress signals and mediate reprogramming of stress responsive genes which ultimately leads to plant defense.

Ectopic expression of a stress-inducible glycosyltransferase from saffron enhances salt and oxidative stress tolerance in Arabidopsis while alters anchor root formation.

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.

New target carotenoids for CCD4 enzymes are revealed with the characterization of a novel stress-induced carotenoid cleavage dioxygenase gene from Crocus sativus.

Apocarotenoid compounds play diverse communication functions in plants, some of them being as hormones, pigments and volatiles. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid cleavage dioxygenase (CCD). The CCD4 family is the largest family of plant CCDs, only present in flowering plants, suggesting a functional diversification associated to the adaptation for specific physiological capacities unique to them. In saffron, two CCD4 genes have been previously isolated from the stigma tissue and related with the generation of specific volatiles involved in the attraction of pollinators. The aim of this study was to identify additional CCD4 members associated with the generation of other carotenoid-derived volatiles during the development of the stigma. The expression of CsCCD4c appears to be restricted to the stigma tissue in saffron and other Crocus species and was correlated with the generation of megastigma-4,6,8-triene. Further, CsCCD4c was up-regulated by wounding, heat, and osmotic stress, suggesting an involvement of its apocarotenoid products in the adaptation of saffron to environmental stresses. The enzymatic activity of CsCCD4c was determined in vivo in Escherichia coli and subsequently in Nicotiana benthamiana by analyzing carotenoids by HPLC-DAD and the volatile products by GC/MS. ß-Carotene was shown to be the preferred substrate, being cleaved at the 9,10 (9',10') bonds and generating ß-ionone, although ß-cyclocitral resulting from a 7,8 (7',8') cleavage activity was also detected at lower levels. Lutein, neoxanthin and violaxanthin levels in Nicotiana leaves were markedly reduced when CsCCD4c is over expressed, suggesting that CsCCD4c recognizes these carotenoids as substrates.

Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis.

Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7',8' double bonds adjacent to a 3-OH-ß-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-ß-apo-8'-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-ß-apo-8'-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the ß-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

Apical dominance in saffron and the involvement of the branching enzymes CCD7 and CCD8 in the control of bud sprouting.

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 non-grass 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.

Characterization of a glucosyltransferase enzyme involved in the formation of kaempferol and quercetin sophorosides in Crocus sativus.

UGT707B1 is a new glucosyltransferase isolated from saffron (Crocus sativus) that localizes to the cytoplasm and the nucleus of stigma and tepal cells. UGT707B1 transcripts were detected in the stigma tissue of all the Crocus species analyzed, but expression analysis of UGT707B1 in tepals revealed its absence in certain species. The analysis of the glucosylated flavonoids present in Crocus tepals reveals the presence of two major flavonoid compounds in saffron: kaempferol-3-O-ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranoside and quercetin-3-O-ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranoside, both of which were absent from the tepals of those Crocus species that did not express UGT707B1. Transgenic Arabidopsis (Arabidopsis thaliana) plants constitutively expressing UGT707B1 under the control of the cauliflower mosaic virus 35S promoter have been constructed and their phenotype analyzed. The transgenic lines displayed a number of changes that resembled those described previously in lines where flavonoid levels had been altered. The plants showed hyponastic leaves, a reduced number of trichomes, thicker stems, and flowering delay. Levels of flavonoids measured in extracts of the transgenic plants showed changes in the composition of flavonols when compared with wild-type plants. The major differences were observed in the extracts from stems and flowers, with an increase in 3-sophoroside flavonol glucosides. Furthermore, a new compound not detected in ecotype Columbia wild-type plants was detected in all the tissues and identified as kaempferol-3-O-sophoroside-7-O-rhamnoside. These data reveal the involvement of UGT707B1 in the biosynthesis of flavonol-3-O-sophorosides and how significant changes in flavonoid homeostasis can be caused by the overproduction of a flavonoid-conjugating enzyme.

Isolation, purification, crystallization and preliminary crystallographic studies of a chitinase from Crocus vernus.

A chitinase has been isolated and purified from Crocus vernus corms. N-terminal amino-acid sequence analysis of the approximately 30 kDa protein showed 33% identity to narbonin, a seed protein from Vicia narbonensis L. The C. vernus chitinase was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 as the main precipitant. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=172.3, b=37.1, c=126.4 Å, ß=127° and two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.1 Å.

Genomic analysis and gene structure of the plant carotenoid dioxygenase 4 family: a deeper study in Crocus sativus and its allies.

The plastoglobule-targeted enzyme carotenoid cleavage dioxygenase (CCD4) mediates the formation of volatile C13 ketones, such as ß-ionone, by cleaving the C9-C10 and C9'-C10' double bonds of cyclic carotenoids. Here, we report the isolation and analysis of CCD4 genomic DNA regions in Crocus sativus. Different CCD4 alleles have been identified: CsCCD4a which is found with and without an intron and CsCCD4b that showed the presence of a unique intron. The presence of different CCD4 alleles was also observed in other Crocus species. Furthermore, comparison of the locations of CCD4 introns within the coding region with CCD4 genes from other plant species suggests that independent gain/losses have occurred. The comparison of the promoter region of CsCCD4a and CsCCD4b with available CCD4 gene promoters from other plant species highlighted the conservation of cis-elements involved in light response, heat stress, as well as the absence and unique presence of cis-elements involved in circadian regulation and low temperature responses, respectively. Functional characterization of the Crocus sativus CCD4a promoter using Arabidopsis plants stably transformed with a DNA fragment of 1400 base pairs (P-CsCCD4a) fused to the ß-glucuronidase (GUS) reporter gene showed that this sequence was sufficient to drive GUS expression in the flower, in particular high levels were detected in pollen.