Carotenoids are isoprenoid pigments synthesized by all photosynthetic organisms and some nonphotosynthetic bacteria and fungi. In plants, carotenoids are essential in protecting the photosynthetic apparatus from photo-oxidation, and represent essential constituents of the light-harvesting and of the reaction center complexes [1][2][3][4]. Carotenoids are also the source of apocarotenoids [5][6][7], which are physiologically active compounds, including the ubiquitous chromophore retinal, the chordate morphogen retinoic acid and the phytohormone abscisic acid as the best-known examples. Further carotenoid-derived signaling molecules are represented by strigolactones, a group of C 15 apocarotenoids attracting both symbiotic arbuscular mycorrhizal fungi and parasitic plants [8,9] and, as recently shown, exerting functions as novel plant hormones regulating shoot branching [10,11]. In addition, the development of arbuscular mycorrhiza is also accompanied by accumulation of cyclohexenone (C 13 ) and mycorradicin (C 14 ) derivatives [12], all of which are apocarotenoids conferring a yellow pigmentation to infected roots [13]. Apocarotenoids, such as bixin in Bixa orellana [14] and saffron in Crocus sativus [15], are plant pigments of economic value.The synthesis of apocarotenoids is initiated by the oxidative cleavage of double bonds in carotenoid Carotenoid cleavage products -apocarotenoids -include biologically active compounds, such as hormones, pigments and volatiles. Their biosynthesis is initiated by the oxidative cleavage of C-C double bonds in carotenoid backbones, leading to aldehydes and ⁄ or ketones. This step is catalyzed by carotenoid oxygenases, which constitute an ubiquitous enzyme family, including the group of plant carotenoid cleavage dioxygenases 1 (CCD1s), which mediates the formation of volatile C 13 ketones, such as b-ionone, by cleaving the C9-C10 and C9¢-C10¢ double bonds of cyclic and acyclic carotenoids. Recently, it was reported that plant CCD1s also act on the C5-C6 ⁄ C5¢-C6¢ double bonds of acyclic carotenes, leading to the volatile C 8 ketone 6-methyl-5-hepten-2-one. Using in vitro and in vivo assays, we show here that rice CCD1 converts lycopene into the three different volatiles, pseudoionone, 6-methyl-5-hepten-2-one, and geranial (C 10 ), suggesting that the C7-C8 ⁄ C7¢-C8¢ double bonds of acyclic carotenoid ends constitute a novel cleavage site for the CCD1 plant subfamily. The results were confirmed by HPLC, LC-MS and GC-MS analyses, and further substantiated by in vitro incubations with the monocyclic carotenoid 3-OH-c-carotene and with linear synthetic substrates. Bicyclic carotenoids were cleaved, as reported for other plant CCD1s, at the C9-C10 and C9¢-C10¢ double bonds. Our study reveals a novel source for the widely occurring plant volatile geranial, which is the cleavage of noncyclic ends of carotenoids.Abbreviations CCD, carotenoid cleavage dioxygenase; GST, glutathione S-transferase;

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.

HighlightsThe substrate specificity and products of tomato carotenoid cleavage dioxygenases 1A and 1B were analyzed.New cleavage sites leading to new enzymatic products were identified.CCD1A and 1B link the carotenoid pattern of tomatoes to the many isoprenoid volatiles produced.

Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.

SummaryMonoterpenes are important constituents of the aromas of food and beverages, including wine. Among monoterpenes in wines, wine lactone has the most potent odor. It was proposed to form via acid-catalyzed cyclization of (E)-8-carboxylinalool during wine maturation. It only reaches very low concentrations in wine but its extremely low odor detection threshold makes it an important aroma compound.Using LC-MS/MS, we show here that the (E)-8-carboxylinalool content in wines correlates with their wine lactone content and estimate the kinetic constant for the very slow formation of wine lactone from (E)-8-carboxylinalool. We show that (E)-8-carboxylinalool is accumulated as a glycoside in grape (Vitis vinifera) berries and that one of the cytochrome P450 enzymes most highly expressed in maturing berries, CYP76F14, efficiently oxidizes linalool to (E)-8-carboxylinalool.Our analysis of (E)-8-carboxylinalool in Riesling 9 Gewurztraminer grapevine progeny established that the CYP76F14 gene co-locates with a quantitative trait locus for (E)-8-carboxylinalool content in grape berries.Our data support the role of CYP76F14 as the major (E)-8-carboxylinalool synthase in grape berries and the role of (E)-8-carboxylinalool as a precursor to wine lactone in wine, providing new insights into wine and grape aroma metabolism, and new methods for food and aroma research and production.