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Carotenoids are the precursors of important fragrance compounds in flowers of Osmanthus fragrans Lour. var. aurantiacus, which exhibit the highest diversity of carotenoid-derived volatiles among the flowering plants investigated. A cDNA encoding a carotenoid cleavage enzyme, OfCCD1, was identified from transcripts isolated from flowers of O. fragrans Lour. It is shown that the recombinant enzymes cleave carotenes to produce α-ionone and β-ionone in in vitro assays. It was also found that carotenoid content, volatile emissions, and OfCCD1 transcript levels are subjected to photorhythmic changes and principally increased during daylight hours. At the times when OfCCD1 transcript levels reached their maxima, the carotenoid content remained low or slightly decreased. The emission of ionones was also higher during the day; however, emissions decreased at a lower rate than the transcript levels. Moreover, carotenoid content increased from the first to the second day, whereas the volatile release decreased, and the OfCCD1 transcript levels displayed steady-state oscillations, suggesting that the substrate availability in the cellular compartments is changing or other regulatory factors are involved in volatile norisoprenoid formation. Furthermore, the sensory evaluation of the aroma of the model mixtures suggests that the proportionally higher contribution of α-ionone and β-ionone to total volatile emissions in the evening is probably the reason for the increased perception by humans of the scent emission of Osmanthus flowers.

The sweet osmanthus carotenoid cleavage dioxygenase 4 (OfCCD4) cleaves carotenoids such as β-carotene and zeaxanthin to yield β-ionone. OfCCD4 is a member of the CCD gene family, and its promoter contains a W-box palindrome with two reversely oriented TGAC repeats, which are the proposed binding sites of WRKY transcription factors. We isolated three WRKY cDNAs from the petal of Osmanthus fragrans . One of them, OfWRKY3, encodes a protein containing two WRKY domains and two zinc finger motifs. OfWRKY3 and OfCCD4 had nearly identical expression profile in petals of ‘Dangui’ and ‘Yingui’ at different flowering stages and showed similar expression patterns in petals treated by salicylic acid, jasmonic acid and abscisic acid. Activation of OfCCD4 pro :GUS by OfWRKY3 was detected in coinfiltrated tobacco leaves and very weak GUS activity was detected in control tissues, indicating that OfWRKY3 can interact with the OfCCD4 promoter. Yeast one-hybrid and electrophoretic mobility shift assay showed that OfWRKY3 was able to bind to the W-box palindrome motif present in the OfCCD4 promoter. These results suggest that OfWRKY3 is a positive regulator of the OfCCD4 gene, and might partly account for the biosynthesis of β-ionone in sweet osmanthus.

Early domestication and extensive cultivation have meant that staple Mediterranean fruit crops such as olives, grapes and dates exist in wild-looking forms that are secondary derivatives produced by sexual reproduction among cultivated plants (cultivars), which were initially propagated vegetatively. By using genetic markers associated with characters that render plants unsuitable for domestication, we show here that genuinely wild olive trees, which cannot be distinguished morphologically from feral forms, still survive in a few Mediterranean forests. These wild stocks are genetically distinct and more variable than either the crop strains or their derived feral forms, a finding that has important implications for the conservation of these ancient lineages.

Endo-β-1,3-glucanases are widespread enzymes with glycosyl hydrolitic activity involved in carbohydrate remodelling during the germination and pollen tube growth. Although members of this protein family with allergenic activity have been reported, their effective contribution to allergy is little known. In this work, we identified Fra e 9 as a novel allergenic β-1,3-glucanase from ash pollen. We produced the catalytic and carbohydrate-binding domains as two independent recombinant proteins and characterized them from structural, biochemical and immunological point of view in comparison to their counterparts from olive pollen. We showed that despite having significant differences in biochemical activity Fra e 9 and Ole e 9 display similar IgE-binding capacity, suggesting that β-1,3-glucanases represent an heterogeneous family that could display intrinsic allergenic capacity. Specific cDNA encoding Fra e 9 was cloned and sequenced. The full-length cDNA encoded a polypeptide chain of 461 amino acids containing a signal peptide of 29 residues, leading to a mature protein of 47760.2 Da and a pI of 8.66. An N-terminal catalytic domain and a C-terminal carbohydrate-binding module are the components of this enzyme. Despite the phylogenetic proximity to the olive pollen β-1,3-glucanase, Ole e 9, there is only a 39% identity between both sequences. The N- and C-terminal domains have been produced as independent recombinant proteins in Escherichia coli and Pichia pastoris, respectively. Although a low or null enzymatic activity has been associated to long β-1,3-glucanases, the recombinant N-terminal domain has 200-fold higher hydrolytic activity on laminarin than reported for Ole e 9. The C-terminal domain of Fra e 9, a cysteine-rich compact structure, is able to bind laminarin. Both molecules retain comparable IgE-binding capacity when assayed with allergic sera. In summary, the structural and functional comparison between these two closely phylogenetic related enzymes provides novel insights into the complexity of β-1,3-glucanases, representing a heterogeneous protein family with intrinsic allergenic capacity.