Functional effect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat flavour formation

Grape berries of Muscat cultivars (Vitis vinifera L.) contain high levels of monoterpenols and exhibit a distinct aroma related to this composition of volatiles. A structural gene of the plastidial methyl-erythritol-phosphate (MEP) pathway, 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS), was recently suggested as a candidate gene for this trait, having been co-localized with a major quantitative trait locus for linalool, nerol, and geraniol concentrations in berries. In addition, a structured association study discovered a putative causal single nucleotide polymorphism (SNP) responsible for the substitution of a lysine with an asparagine at position 284 of the VvDXS protein, and this SNP was significantly associated with Muscat-flavoured varieties. The significance of this nucleotide difference was investigated by comparing the monoterpene profiles with the expression of VvDXS alleles throughout berry development in Moscato Bianco, a cultivar heterozygous for the SNP mutation. Although correlation was detected between the VvDXS transcript profile and the accumulation of free monoterpenol odorants, the modulation of VvDXS expression during berry development appears to be independent of nucleotide variation in the coding sequence. In order to assess how the non-synonymous mutation may enhance Muscat flavour, an in vitro characterization of enzyme isoforms was performed followed by in vivo overexpression of each VvDXS allele in tobacco. The results showed that the amino acid non-neutral substitution influences the enzyme kinetics by increasing the catalytic efficiency and also dramatically affects monoterpene levels in transgenic lines. These findings confirm a functional effect of the VvDXS gene polymorphism and may pave the way for metabolic engineering of terpenoid contents in grapevine

NPBTs FOR SUSTAINABLE VITICULTURE MANAGEMENT TO BIOTIC AND ABIOTIC STRESS

New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for plant improvement to biotic and abiotic stresses satisfying the European Policies requirements that promote chemical input reduction and a more sustainable agriculture. We decided to apply genome editing (via CRISPR/Cas9) focusing on susceptibility genes to control powdery mildew: we chosen to knock-out two genes belonging to MLO (Mildew Locus O) family: VvMLO7 and VvMLO6. The same approach was used to cope with abiotic stresses, in specifc drought, performing a knock-out of four genes, two belonging to GST (Glutathione S-Transferase) and two to PME (Pectin Methyl Esterase) gene families. In parallel to genome editing, we also applied cisgenesis to move the resistance locus RPV3-1 (Resistance to Plasmopara viticola) into economically important cultivars. This locus is formed by two di\ufb00erent genes that were inserted individually and in combination to evaluate their e\ufb00ects. One of the drawbacks linked to classical Agrobacterium tumefaciens mediated transformation is the insertion of unrelated transgene (e.g., antibiotic resistance). These markers are required for transgenic plants selection, but undesirable to be retained in commercial plants due to possible toxicity or allergenicity to humans and animals, in addition to their potential hazards for the environment. To overcome these limits, we exploit an inducible excision system based on a Cre-lox recombinase technology controlled by a heat-shock inducible promoter that will be activated once the transformation event(s) will be confrmed. Embryogenic calli of Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese, were used in stable transformation with A. tumefaciens carrying the genome editing construct with the MLO-guideRNAs and the cisgenic construct carrying the two RPV3-1 genes. Embryogenic calli of rootstocks 110 Richter and SO4 were transformed with genome editing construct carrying GST and PME guideRNAs in two independent transformations. Regenerated embryos from all the transformation events are now under evaluation

New plant breeding technologies towards a more sustainable viticulture

European grapevine cultivars are highly susceptible to many pathogens that are managed through large pesticide use. Nevertheless, the European policies promote pesticide use reduction and new environmentally friendly methods for a more sustainable agriculture. In this framework, grapevine genetic improvement could benefit from New Plant Breeding Technologies. In order to reduce fungal susceptibility, we will produce knock-out plants from embryogenic calli using CRISPR/Cas9 technology. Studies in barley reported the acquisition of powdery mildew resistance by knocking out susceptibility genes belonging to the MLO (Mildew Locus O) family. In this study, our approach takes advantage from CRISPR/Cas9 technology to perform a multiple knockout of MLO genes. Among the 17 VvMLOs reported in grapevine we designed constructs to target VvMLO6 and VvMLO7. Golden Gate assembly was used to produce three different constructs (containing two guideRNAs for each gene) to knocking-out the targets singularly or by producing a double mutant. Usually, the genetic engineering techniques, mediated by A. tumefaciens, involve the insertion of exogenous selectable marker genes. These markers are required for selection of transgenic plants, but they are undesirable to be retained in commercial transgenic plants due to possible toxicity or allergenicity to humans and potential environmental hazard. To overcome these limits, we opted for a \u201cclean\u201d editing strategy developing an inducible excision system. This approach is based on a recombinase technology involving the Cre-loxP system from the P1 bacteriophage under a heat-shock inducible promoter to be activated once the editing event(s) will be confirmed. Obtainment of embryogenic calli is one of the main bottlenecks for application of CRISPR/Cas9: for two seasons, we collected inflorescences from Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese cultivars and two rootstocks, 110 Richter and SO4, cultured and maintained in vitro up to embryo development and then used to perform Agrobacterium tumefaciens GV3101 mediated transformation

Novel functional microRNAs from virus-free and infected Vitis vinifera plants under water stress

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the post-transcriptional control of several pathway intermediates, thus playing pivotal roles in plant growth, development and response to biotic and abiotic stresses. In recent years, the grapevine genome release, small(s)-RNAseq and degradome-RNAseq together has allowed the discovery and characterisation of many miRNA species, thus rendering the discovery of additional miRNAs difficult and uncertain. Taking advantage of the miRNA responsiveness to stresses and the availability of virus-free Vitis vinifera plants and those infected only by a latent virus, we have analysed grapevines subjected to drought in greenhouse conditions. The sRNA-seq and other sequence-specific molecular analyses have allowed us to characterise conserved miRNA expression profiles in association with specific eco-physiological parameters. In addition, we here report 12 novel grapevine-specific miRNA candidates and describe their expression profile. We show that latent viral infection can influence the miRNA profiles of V. vinifera in response to drought. Moreover, study of eco-physiological parameters showed that photosynthetic rate, stomatal conductance and hydraulic resistance to water transport were significantly influenced by drought and viral infection. Although no unequivocal cause–effect explanation could be attributed to each miRNA target, their contribution to the drought response is discussed

Editorial: Functional Genomics in Fruit Trees: From 'Omics to Sustainable Biotechnologies

This Research Topic of Frontiers in Plant Science collects 8 manuscripts, focused on fruit crops of high commercial interest, such as bayberry, blueberry, pear, grapevine, citrus, and walnut. The breadth of solutions and approaches that omics offer us today, and the applications and perspectives that are looming over a short time horizon, are illustrated with a particular focus on fruit qualitative traits, related to firmness and post-harvest (Cappai et al.), ripening (Honaas et al.), and lignin accumulation (Cao et al.), on improving knowledge on secondary metabolites, such as phenolic compounds (Saxe et al.), as well as on investigating earliest responses to pathogens (Wei et al.). Moreover, the Research Topic illustrates a technological application path from whole-genome sequencing (Wu et al.) to the resequencing (Tanaka et al.), to achieve the ultimate objective of modulating genes using the New Breeding Techniques (Salonia et al.)