SummaryPetunia hybrida W115 was transformed with a Clarkia breweri S-linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S-linalool produced by the plant in the various tissues is not present as free linalool, but was ef®ciently converted to non-volatile S-linalyl-b-Dglucopyranoside by the action of endogenous glucosyltransferase. The results presented demonstrate that monoterpene production can be altered by genetic modi®cation, and that the compounds produced can be converted by endogenous enzymatic activity.

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Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting ␤-pinene, limonene, and ␥-terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes.Plants are producing a wide range of different volatile metabolites by different biosynthetic pathways, each having their own complex regulation (Dudareva and Pichersky, 2000). The regulated emission of volatiles from flowers has been suggested to correlate with the patterns of activity of the organisms that are interacting with the plant such as pollinators, herbivores, and predators of the herbivores (Kolosova et al., 2001;Pichersky and Gershenzon, 2002). Emission of volatiles was shown to be regulated at the gene expression level and often displays a nocturnal or diurnal circadian rhythm as was shown for methylbenzoate emission of snapdragon (Antirrhinum majus), tobacco, and Clarkia breweri (Dudareva et al., 1996(Dudareva et al., , 2000 Kolosova et al., 2001).Terpenoid synthases are to a large extent responsible for the diversity of terpenoid structures involved in floral scent or in herbivore induced volatiles (Bohlmann et al., 1998). Monoterpenes are C 10 compounds, synthesized from geranyldiphosphate (GPP) via the plastid localized 2C-methyl-derythritol-4-phosphate (MEP) pathway (Eisenreich et al., 1997). They are of high economic value because they are products extensively used as flavor and fragrance additives in food and cosmetics (Verlet, 1993). Furthermore, they are of interest for their medicinal, e.g. anticarcinogenic, properties (Crowell, 1999). Their biological significance lies in their involvement in plant-insect, plant-pathogen, and plantplant interactions (Wink, 1999). Monoterpenes can be released in a diurnal rhythm as was shown for rose flowers (Helsper et al., 1998(Helsper et al., , 2001. A monoterpene synthase mRNA from Artemisia annua has recently been shown to be regulated in a circadian rhythm, and the expression profile was shown to correlate with the content and emission of the monoterpene produced by the corresponding enzyme (Lu et al., 2002). Monoterpene emission is also affected by environmental conditions such as temperature and light (Schuh et al., 1997). Tobacco varieties have also been investigated for their emission profiles and were shown to emit mono-and sesquiterpenes. Emission of volatiles from leaves was 30-to 100-fold lower than e...

BackgroundiTRAQ is a proteomics technique that uses isobaric tags for relative and absolute quantitation of tryptic peptides. In proteomics experiments, the detection and high confidence annotation of proteins and the significance of corresponding expression differences can depend on the quality and the species specificity of the tryptic peptide map database used for analysis of the data. For species for which finished genome sequence data are not available, identification of proteins relies on similarity to proteins from other species using comprehensive peptide map databases such as the MSDB.ResultsWe were interested in characterizing ripening initiation ('veraison') in grape berries at the protein level in order to better define the molecular control of this important process for grape growers and wine makers. We developed a bioinformatic pipeline for processing EST data in order to produce a predicted tryptic peptide database specifically targeted to the wine grape cultivar, Vitis vinifera cv. Cabernet Sauvignon, and lacking truncated N- and C-terminal fragments. By searching iTRAQ MS/MS data generated from berry exocarp and mesocarp samples at ripening initiation, we determined that implementation of the custom database afforded a large improvement in high confidence peptide annotation in comparison to the MSDB. We used iTRAQ MS/MS in conjunction with custom peptide db searches to quantitatively characterize several important pathway components for berry ripening previously described at the transcriptional level and confirmed expression patterns for these at the protein level.ConclusionWe determined that a predicted peptide database for MS/MS applications can be derived from EST data using advanced clustering and trimming approaches and successfully implemented for quantitative proteome profiling. Quantitative shotgun proteome profiling holds great promise for characterizing biological processes such as fruit ripening initiation and may be further improved by employing preparative techniques and/or analytical equipment that increase peptide detection sensitivity via a shotgun approach.

Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were (+)-limonene (two cDNAs) (-)-b-pinene and c-terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn 2+ as bivalent metal ion cofactor resulted in higher activity than Mg 2+ , with an optimum concentration of 0.6 mM. K m values ranged from 0.7 to 3.1 lM. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90% of the main components present.