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.

Identification and characterization of apocarotenoid modifiers and carotenogenic enzymes for biosynthesis of crocins in Buddleja davidii flowers

Crocetin biosynthesis in Buddleja davidii flowers proceeds through a zeaxanthin cleavage pathway catalyzed by two carotenoid cleavage dioxygenases (BdCCD4.1 and BdCCD4.3), followed by oxidation and glucosylation reactions that lead to the production of crocins. We isolated and analyzed the expression of 12 genes from the carotenoid pathway in B. davidii flowers and identified four candidate genes involved in the biosynthesis of crocins (BdALDH, BdUGT74BC1, BdUGT74BC2, and BdUGT94AA3). In addition, we characterized the profile of crocins and their carotenoid precursors, following their accumulation during flower development. Overall, seven different crocins, crocetin, and picrocrocin were identified in this study. The accumulation of these apocarotenoids parallels tissue development, reaching the highest concentration when the flower is fully open. Notably, the pathway was regulated mainly at the transcript level, with expression patterns of a large group of carotenoid precursor and apocarotenoid genes (BdPSY2, BdPDS2, BdZDS, BdLCY2, BdBCH, BdALDH, and BdUGT Genes) mimicking the accumulation of crocins. Finally, we used comparative correlation network analysis to study how the synthesis of these valuable apocarotenoids diverges among B. davidii, Gardenia jasminoides, and Crocus sativus, highlighting distinctive differences which could be the basis of the differential accumulation of crocins in the three species.

New floristic records from Central Europe 8 (reports 109-121)

The presented eighth part of the series includes thirteen new chorological records of vascular plants, one from Hungary, six from Poland and six from Slovakia. In Hungary, Ventenata dubia is reported. In Poland, two native taxa Bolboschoenus planiculmis and Najas marina subsp. marina and four alien taxa Buddleja davidii, Lupinus ehrenbergii var. ehrenbergii, Miscanthus sacchariflorus and Sedum sarmentosum are reported. In Slovakia, two native taxa Taraxacum paucilobum with distribution map and Cotoneaster integerrimus are reported as well as four alien taxa Azolla filiculoides, Eichhornia crassipes, Euphorbia prostrata and Pistia stratioites.

The Selection of Ornamental Plant for Landscape Design of Pollination Garden at Bogor Botanic Gardens

Bogor Botanic Gardens is one of the outstanding green areas in Bogor City. Thousands of plant species are growing and providing suitable habitats and feed for various pollinators, such as butterflies, bees, and wasps. A well-designed pollination garden will benefit the pollinators and also create an educational facility for the community. This study aimed to select and characterize species of flowering plants that effectively attract pollinators, particularly Lepidoptera and Hymenoptera, in the pollination garden candidate area. The observa-tion had been carried out on 12 ornamental plant species: Pennisetum purpureum, Cosmos sp., Pachystachys lutea, Cuphea hyssopifolia, Orthosiphon aristatus, Lantana camara, Citrus aurantifolia, Buddleja davidii, Ixora javanica, Nerium oleander, Hibiscus rosa-sinensis, and Aglaia odorata. The method for observing pollinators was a standard walking method. Pollinator photos, its number, its visit time, and the plants it visited were recorded. A total of 23 species of Lepidoptera (20 species from 4 families and 3 species were unidentified) and 9 species of Hymenoptera (4 families) were found in the area with the best obser-vation time at 08.30-09.30 (GMT+7). The most visited plants by pollinators were Cuphea hyssopifolia, Lantana camara and Orthosiphon aristatus. Lepidoptera and Hymenoptera preferred blue-purple and bright-orange-yellow flowers. In addition, the flower characters that might influence pollinator visits are also described. The result would be an essential recommendation for plant selection, landscape design, and construction of the pollination garden in Bogor Botanic Gardens.

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

ABSTRACTCrocins 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.

Alien invasive plants in Belgian limestone quarries

Description of the subject. Vegetation of high conservation value can establish in quarries, during or after exploitation. Alien plants could hamper this process and cause additional rehabilitation costs. However, the situation of plant invasion in quarries is largely unknown. Objectives. We aimed to assess alien plant invasion in active and abandoned quarries, and to identify the most invaded quarry sectors. Method. We surveyed 6,692 plots in 31 quarries in Belgium and recorded occurrence, density and cover of the 65 listed alien invasive plants in Belgium. Results. Fourteen species were recorded and 25 quarries contained at least one species. The two most occurring species, Buddleja davidii Franch. and Senecio inaequidens DC., were more widespread in quarries in activity. All sectors of the quarries were affected by alien plant invasion. Conclusions. Alien plant invasion in limestone quarries is highly variable, but significant. Considering the ecological potential of quarry sites, this issue should be better tackled.