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Despite a consolidated increase in their employment, herbicide-tolerant (HT) and insect-resistant (IR) GM varieties have commonly been suspected to represent a threat to biodiversity. In this chapter, we analyze the major concerns related to the potential persistence and invasiveness of GM plants, selection of superweeds and resistant insects, effects on non-target organisms including vertical and horizontal gene flow, with the acquisition of antibiotic resistance and biological contamination. Mitigation measures to reduce the ecological impact on biodiversity are briefly considered.

Terpenoids, especially monoterpenes, are major aroma-impact compounds in grape and wine. Previous studies highlighted a key regulatory role for grapevine 1-deoxy-D-xylulose 5-phosphate synthase 1 (VvDXS1), the first enzyme of the methylerythritol phosphate pathway for isoprenoid precursor biosynthesis. Here, the parallel analysis of VvDXS1 genotype and terpene concentration in a germplasm collection demonstrated that VvDXS1 sequence has a very high predictive value for the accumulation of monoterpenes and also has an influence on sesquiterpene levels. A metabolic engineering approach was applied by expressing distinct VvDXS1 alleles in the grapevine model system "microvine" and assessing the effects on downstream pathways at transcriptional and metabolic level in different organs and fruit developmental stages. The underlying goal was to investigate two potential perturbation mechanisms, the former based on a significant over-expression of the wild-type (neutral) VvDXS1 allele and the latter on the ex-novo expression of an enzyme with increased catalytic efficiency from the mutated (muscat) VvDXS1 allele. The integration of the two VvDXS1 alleles in distinct microvine lines was found to alter the expression of several terpenoid biosynthetic genes, as assayed through an ad hoc developed TaqMan array based on cDNA libraries of four aromatic cultivars. In particular, enhanced transcription of monoterpene, sesquiterpene and carotenoid pathway genes was observed. The accumulation of monoterpenes in ripe berries was higher in the transformed microvines compared to control plants. This effect is predominantly attributed to the improved activity of the VvDXS1 enzyme coded by the muscat allele, whereas the up-regulation of VvDXS1 plays a secondary role in the increase of monoterpenes.

Monoterpenes confer typical floral notes to ‘Muscat’ grapevine varieties and, to a lesser extent, to other aromatic non-Muscat varieties. Previous studies have led to the identification and functional characterization of some enzymes and genes in this pathway. However, the underlying genetic map is still far from being complete. For example, the specific steps of monoterpene metabolism and its regulation are largely unknown. With the aim of identifying new candidates for the missing links, we applied an integrative functional genomics approach based on the targeted metabolic and genome-wide transcript profiling of Moscato Bianco ripening berries. In particular, gas chromatography-mass spectrometry analysis of free and bound terpenoid compounds was combined with microarray analysis in the skins of berries collected at five developmental stages from pre-veraison to over-ripening. Differentially expressed metabolites and probes were identified in the pairwise comparison between time points by using the early stage as a reference. Metabolic and transcriptomic data were integrated through pairwise correlation and clustering approaches to discover genes linked with particular metabolites or groups of metabolites. These candidate transcripts were further checked for co-localization with quantitative trait loci (QTLs) affecting aromatic compounds. Our findings provide valuable insights into the biological networks of grapevine secondary metabolism, both at the catalytic and regulatory levels. Examples include a nudix hydrolase as component of a terpene synthase-independent pathway for monoterpene biosynthesis, genes potentially involved in monoterpene metabolism (cytochrome P450 hydroxylases, epoxide hydrolases, glucosyltransferases), transport (vesicle-associated proteins, ABCG transporters, glutathione S-transferases, amino acid permeases), and transcriptional control (transcription factors of the ERF, MYB and NAC families, intermediates in light- and circadian cycle-mediated regulation with supporting evidence from the literature and additional regulatory genes with a previously unreported association to monoterpene accumulation).