Prostaglandin D2 synthase controls Schwann cells metabolism.

We previously reported that in the absence of Prostaglandin D2 synthase (L-PGDS) peripheral nerves are hypomyelinated in development and that with aging they present aberrant myelin sheaths. We now demonstrate that L-PGDS expressed in Schwann cells is part of a coordinated program aiming at preserving myelin integrity. In vivo and in vitro lipidomic, metabolomic and transcriptomic analyses confirmed that myelin lipids composition, Schwann cells energetic metabolism and key enzymes controlling these processes are altered in the absence of L-PGDS. Moreover, Schwann cells undergo a metabolic rewiring and turn to acetate as the main energetic source. Further, they produce ketone bodies to ensure glial cell and neuronal survival. Importantly, we demonstrate that all these changes correlate with morphological myelin alterations and describe the first physiological pathway implicated in preserving PNS myelin. Collectively, we posit that myelin lipids serve as a reservoir to provide ketone bodies, which together with acetate represent the adaptive substrates Schwann cells can rely on to sustain the axo-glial unit and preserve the integrity of the PNS.

Dissecting the effect of soil on plant phenology and berry transcriptional plasticity in two Italian grapevine varieties (Vitis vinifera L.).

Grapevine embodies a fascinating species as regards phenotypic plasticity and genotype-per-environment interactions. The terroir, namely the set of agri-environmental factors to which a variety is subjected, can influence the phenotype at the physiological, molecular, and biochemical level, representing an important phenomenon connected to the typicality of productions. We investigated the determinants of plasticity by conducting a field-experiment where all terroir variables, except soil, were kept as constant as possible. We isolated the effect of soils collected from different areas, on phenology, physiology, and transcriptional responses of skin and flesh of a red and a white variety of great economic value: Corvina and Glera. Molecular results, together with physio-phenological parameters, suggest a specific effect of soil on grapevine plastic response, highlighting a higher transcriptional plasticity of Glera in respect to Corvina and a marked response of skin compared to flesh. Using a novel statistical approach, we identified clusters of plastic genes subjected to the specific influence of soil. These findings could represent an issue of applicative value, posing the basis for targeted agricultural practices to enhance the desired characteristics for any soil/cultivar combination, to improve vineyards management for a better resource usage and to valorize vineyards uniqueness maximizing the terroir-effect.

Physiological and Transcriptomic Evaluation of Drought Effect on Own-Rooted and Grafted Grapevine Rootstock (1103P and 101-14MGt).

Grapevines worldwide are grafted onto Vitis spp. rootstocks in order to improve their tolerance to biotic and abiotic stresses. Thus, the response of vines to drought is the result of the interaction between the scion variety and the rootstock genotype. In this work, the responses of genotypes to drought were evaluated on 1103P and 101-14MGt plants, own-rooted and grafted with Cabernet Sauvignon, in three different water deficit conditions (80, 50, and 20% soil water content, SWC). Gas exchange parameters, stem water potential, root and leaf ABA content, and root and leaf transcriptomic response were investigated. Under well-watered conditions, gas exchange and stem water potential were mainly affected by the grafting condition, whereas under sever water deficit they were affected by the rootstock genotype. Under severe stress conditions (20% SWC), 1103P showed an "avoidance" behavior. It reduced stomatal conductance, inhibited photosynthesis, increased ABA content in the roots, and closed the stomata. The 101-14MGt maintained a high photosynthetic rate, limiting the reduction of soil water potential. This behavior results in a "tolerance" strategy. An analysis of the transcriptome showed that most of the differentially expressed genes were detected at 20% SWC, and more significantly in roots than in leaves. A core set of genes has been highlighted on the roots as being related to the root response to drought that are not affected by genotype nor grafting. Genes specifically regulated by grafting and genes specifically regulated by genotype under drought conditions have been identified as well. The 1103P, more than the 101-14MGt, regulated a high number of genes in both own-rooted and grafted conditions. This different regulation revealed that 1103P rootstock readily perceived the water scarcity and rapidly faced the stress, in agreement with its avoidance strategy.

Distinct and Temporally Stable Assembly Mechanisms Shape Bacterial and Fungal Communities in Vineyard Soils.

Microbial communities in agricultural soils are fundamental for plant growth and in vineyard ecosystems contribute to defining regional wine quality. Managing soil microbes towards beneficial outcomes requires knowledge of how community assembly processes vary across taxonomic groups, spatial scales, and through time. However, our understanding of microbial assembly remains limited. To quantify the contributions of stochastic and deterministic processes to bacterial and fungal assembly across spatial scales and through time, we used 16 s rRNA gene and ITS sequencing in the soil of an emblematic wine-growing region of Italy.Combining null- and neutral-modelling, we found that assembly processes were consistent through time, but bacteria and fungi were governed by different processes. At the within-vineyard scale, deterministic selection and homogenising dispersal dominated bacterial assembly, while neither selection nor dispersal had clear influence over fungal assembly. At the among-vineyard scale, the influence of dispersal limitation increased for both taxonomic groups, but its contribution was much larger for fungal communities. These null-model-based inferences were supported by neutral modelling, which estimated a dispersal rate almost two orders-of-magnitude lower for fungi than bacteria.This indicates that while stochastic processes are important for fungal assembly, bacteria were more influenced by deterministic selection imposed by the biotic and/or abiotic environment. Managing microbes in vineyard soils could thus benefit from strategies that account for dispersal limitation of fungi and the importance of environmental conditions for bacteria. Our results are consistent with theoretical expectations whereby larger individual size and smaller populations can lead to higher levels of stochasticity.

CRISPR/Cas9 genome-editing applied to MdPGT1 in apple results in reduced foliar phloridzin without impacting plant growth.

Phloridzin is the most abundant polyphenolic compound in apple (Malus × domestica Borkh.), which results from the action of a key phloretin-specific UDP-2'-O-glucosyltransferase (MdPGT1). Here, we simultaneously assessed the effects of targeting MdPGT1 by conventional transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing. To this end, we conducted transcriptomic and metabolic analyses of MdPGT1 RNA interference knockdown and genome-edited lines. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology, whereas genome-edited lines exhibited normal growth despite reduced foliar phloridzin. RNA-sequencing analysis identified a common core of regulated genes, involved in phenylpropanoid and flavonoid pathways. However, we identified genes and processes differentially modulated in stunted and genome-edited lines, including key transcription factors and genes involved in phytohormone signalling. Therefore, we conducted a phytohormone profiling to obtain insight into their role in the phenotypes observed. We found that salicylic and jasmonic acid were increased in dwarf lines, whereas auxin and ABA showed no correlation with the growth phenotype. Furthermore, bioactive brassinosteroids were commonly up-regulated, whereas gibberellin GA4 was distinctively altered, showing a sharp decrease in RNA interference knockdown lines. Expression analysis by reverse transcriptase-quantitative polymerase chain reaction expression analysis further confirmed transcriptional regulation of key factors involved in brassinosteroid and gibberellin interaction. These findings suggest that a differential modulation of phytohormones may be involved in the contrasting effects on growth following phloridzin reduction. The present study also illustrates how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple.

Identification of Candidate Genes Associated With Tolerance to Apple Replant Disease by Genome-Wide Transcriptome Analysis.

Apple replant disease (ARD) is a worldwide economic risk in apple cultivation for fruit tree nurseries and fruit growers. Several studies on the reaction of apple plants to ARD are documented but less is known about the genetic mechanisms behind this symptomatology. RNA-seq analysis is a powerful tool for revealing candidate genes that are involved in the molecular responses to biotic stresses in plants. The aim of our work was to find differentially expressed genes in response to ARD in Malus. For this, we compared transcriptome data of the rootstock 'M9' (susceptible) and the wild apple genotype M. ×robusta 5 (Mr5, tolerant) after cultivation in ARD soil and disinfected ARD soil, respectively. When comparing apple plantlets grown in ARD soil to those grown in disinfected ARD soil, 1,206 differentially expressed genes (DEGs) were identified based on a log2 fold change, (LFC) ≥ 1 for up- and ≤ -1 for downregulation (p < 0.05). Subsequent validation revealed a highly significant positive correlation (r = 0.91; p < 0.0001) between RNA-seq and RT-qPCR results indicating a high reliability of the RNA-seq data. PageMan analysis showed that transcripts of genes involved in gibberellic acid (GA) biosynthesis were significantly enriched in the DEG dataset. Most of these GA biosynthesis genes were associated with functions in cell wall stabilization. Further genes were related to detoxification processes. Genes of both groups were expressed significantly higher in Mr5, suggesting that the lower susceptibility to ARD in Mr5 is not due to a single mechanism. These findings contribute to a better insight into ARD response in susceptible and tolerant apple genotypes. However, future research is needed to identify the defense mechanisms, which are most effective for the plant to overcome ARD.

Absence of increased genomic variants in the cyanobacterium Chroococcidiopsis exposed to Mars-like conditions outside the space station.

Despite the increasing interest in using microbial-based technologies to support human space exploration, many unknowns remain not only on bioprocesses but also on microbial survivability and genetic stability under non-Earth conditions. Here the desert cyanobacterium Chroococcidiopsis sp. CCMEE 029 was investigated for robustness of the repair capability of DNA lesions accumulated under Mars-like conditions (UV radiation and atmosphere) simulated in low Earth orbit using the EXPOSE-R2 facility installed outside the International Space Station. Genomic alterations were determined in a space-derivate of Chroococcidiopsis sp. CCMEE 029 obtained upon reactivation on Earth of the space-exposed cells. Comparative analysis of whole-genome sequences showed no increased variant numbers in the space-derivate compared to triplicates of the reference strain maintained on the ground. This result advanced cyanobacteria-based technologies to support human space exploration.

A roadmap for the generation of benchmarking resources for antimicrobial resistance detection using next generation sequencing.

Next Generation Sequencing technologies significantly impact the field of Antimicrobial Resistance (AMR) detection and monitoring, with immediate uses in diagnosis and risk assessment. For this application and in general, considerable challenges remain in demonstrating sufficient trust to act upon the meaningful information produced from raw data, partly because of the reliance on bioinformatics pipelines, which can produce different results and therefore lead to different interpretations. With the constant evolution of the field, it is difficult to identify, harmonise and recommend specific methods for large-scale implementations over time. In this article, we propose to address this challenge through establishing a transparent, performance-based, evaluation approach to provide flexibility in the bioinformatics tools of choice, while demonstrating proficiency in meeting common performance standards. The approach is two-fold: first, a community-driven effort to establish and maintain "live" (dynamic) benchmarking platforms to provide relevant performance metrics, based on different use-cases, that would evolve together with the AMR field; second, agreed and defined datasets to allow the pipelines' implementation, validation, and quality-control over time. Following previous discussions on the main challenges linked to this approach, we provide concrete recommendations and future steps, related to different aspects of the design of benchmarks, such as the selection and the characteristics of the datasets (quality, choice of pathogens and resistances, etc.), the evaluation criteria of the pipelines, and the way these resources should be deployed in the community.

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer.

Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

Two-omics data revealed commonalities and differences between Rpv12- and Rpv3-mediated resistance in grapevine.

Plasmopara viticola is the causal agent of grapevine downy mildew (DM). DM resistant varieties deploy effector-triggered immunity (ETI) to inhibit pathogen growth, which is activated by major resistance loci, the most common of which are Rpv3 and Rpv12. We previously showed that a quick metabolome response lies behind the ETI conferred by Rpv3 TIR-NB-LRR genes. Here we used a grape variety operating Rpv12-mediated ETI, which is conferred by an independent locus containing CC-NB-LRR genes, to investigate the defence response using GC/MS, UPLC, UHPLC and RNA-Seq analyses. Eighty-eight metabolites showed significantly different concentration and 432 genes showed differential expression between inoculated resistant leaves and controls. Most metabolite changes in sugars, fatty acids and phenols were similar in timing and direction to those observed in Rpv3-mediated ETI but some of them were stronger or more persistent. Activators, elicitors and signal transducers for the formation of reactive oxygen species were early observed in samples undergoing Rpv12-mediated ETI and were paralleled and followed by the upregulation of genes belonging to ontology categories associated with salicylic acid signalling, signal transduction, WRKY transcription factors and synthesis of PR-1, PR-2, PR-5 pathogenesis-related proteins.

Novel Aspects on The Interaction Between Grapevine and Plasmopara viticola: Dual-RNA-Seq Analysis Highlights Gene Expression Dynamics in The Pathogen and The Plant During The Battle For Infection.

Mgaloblishvili, a Vitis vinifera cultivar, exhibits unique resistance traits against Plasmopara viticola, the downy mildew agent. This offers the unique opportunity of exploring the molecular responses in compatible and incompatible plant-pathogen interaction. In this study, whole transcriptomes of Mgaloblishvili, Pinot noir (a V. vinifera susceptible cultivar), and Bianca (a resistant hybrid) leaves, inoculated and non-inoculated with the pathogen, were used to identify P. viticola effector-encoding genes and plant susceptibility/resistance genes. Multiple effector-encoding genes were identified in P. viticola transcriptome, with remarkable expression differences in relation to the inoculated grapevine cultivar. Intriguingly, five apoplastic effectors specifically associated with resistance in V. vinifera. Gene coexpression network analysis identified specific modules and metabolic changes occurring during infection in the three grapevine cultivars. Analysis of these data allowed, for the first time, the detection in V. vinifera of a putative P. viticola susceptibility gene, encoding a LOB domain-containing protein. Finally, the de novo assembly of Mgaloblishvili, Pinot noir, and Bianca transcriptomes and their comparison highlighted novel candidate genes that might be at the basis of the resistant phenotype. These results open the way to functional analysis studies and to new perspectives in molecular breeding of grapevine for resistance to P. viticola.

Pseudo-chromosome-length genome assembly of a double haploid "Bartlett" pear (Pyrus communis L.).

BACKGROUND: We report an improved assembly and scaffolding of the European pear (Pyrus communis L.) genome (referred to as BartlettDHv2.0), obtained using a combination of Pacific Biosciences RSII long-read sequencing, Bionano optical mapping, chromatin interaction capture (Hi-C), and genetic mapping. The sample selected for sequencing is a double haploid derived from the same "Bartlett" reference pear that was previously sequenced. Sequencing of di-haploid plants makes assembly more tractable in highly heterozygous species such as P. communis. FINDINGS: A total of 496.9 Mb corresponding to 97% of the estimated genome size were assembled into 494 scaffolds. Hi-C data and a high-density genetic map allowed us to anchor and orient 87% of the sequence on the 17 pear chromosomes. Approximately 50% (247 Mb) of the genome consists of repetitive sequences. Gene annotation confirmed the presence of 37,445 protein-coding genes, which is 13% fewer than previously predicted. CONCLUSIONS: We showed that the use of a doubled-haploid plant is an effective solution to the problems presented by high levels of heterozygosity and duplication for the generation of high-quality genome assemblies. We present a high-quality chromosome-scale assembly of the European pear Pyrus communis and demostrate its high degree of synteny with the genomes of Malus x Domestica and Pyrus x bretschneideri.

Microbiome of vineyard soils is shaped by geography and management.

BACKGROUND: Despite their importance as a reservoir of biodiversity, the factors shaping soil microbial communities and the extent by which these are impacted by cultivation are still poorly understood. Using 16S rRNA gene and ITS sequencing, we characterized the soil microbiota of vineyards and of neighboring permanent grassland soils in the Italian province of Trentino, and correlated their structure and composition to location, chemical properties of the soil, and land management. RESULTS: Bacterial communities had a core of conserved taxa accounting for more than 60% of the reads of each sample, that was influenced both by geography and cultivation. The core fungal microbiota was much smaller and dominated by geography alone. Cultivation altered the structure and composition of the soil microbiota both for bacteria and fungi, with site-specific effects on their diversity. The diversity of bacterial and fungal communities was generally inversely correlated across locations. We identified several taxa that were impacted by the chemical properties and texture of the soil. CONCLUSIONS: Our results highlight the different responses of bacterial and fungal communities to environmental factors and highlight the need to characterize both components of the soil microbiota to fully understand the factors that drive their variability.

Wide transcriptional investigation unravel novel insights of the on-tree maturation and postharvest ripening of 'Abate Fetel' pear fruit.

To decipher the transcriptomic regulation of the on-tree fruit maturation in pear cv. 'Abate Fetel', a RNA-seq transcription analysis identified 8939 genes differentially expressed across four harvesting stages. These genes were grouped into 11 SOTA clusters based on their transcriptional pattern, of which three included genes upregulated while the other four were represented by downregulated genes. Fruit ripening was furthermore investigated after 1 month of postharvest cold storage. The most important variation in fruit firmness, production of ethylene and volatile organic compounds were observed after 5 days of shelf-life at room temperature following cold storage. The role of ethylene in controlling the ripening of 'Abate Fetel' pears was furthermore investigated through the application of 1-methylcyclopropene, which efficiently delayed the progression of ripening by reducing fruit softening and repressing both ethylene and volatile production. The physiological response of the interference at the ethylene receptor level was moreover unraveled investigating the expression pattern of 12 candidate genes, initially selected to validate the RNA-seq profile. This analysis confirmed the effective role of the ethylene competitor in downregulating the expression of cell wall (PG) and ethylene-related genes (ACS, ACO, ERS1, and ERS2), as well as inducing one element involved in the auxin signaling pathway (Aux/IAA), highlighting a possible cross-talk between these two hormones. The expression patterns of these six elements suggest their use as molecular toolkit to monitor at molecular level the progression of the fruit on-tree maturation and postharvest ripening.

PhytoTypeDB: a database of plant protein inter-cultivar variability and function.

Despite a fast-growing number of available plant genomes, available computational resources are poorly integrated and provide only limited access to the underlying data. Most existing databases focus on DNA/RNA data or specific gene families, with less emphasis on protein structure, function and variability. In particular, despite the economic importance of many plant accessions, there are no straightforward ways to retrieve or visualize information on their differences. To fill this gap, we developed PhytoTypeDB (http://phytotypedb.bio.unipd.it/), a scalable database containing plant protein annotations and genetic variants from resequencing of different accessions. The database content is generated by an integrated pipeline, exploiting state-of-the-art methods for protein characterization requiring only the proteome reference sequence and variant calling files. Protein names for unknown proteins are inferred by homology for over 95% of the entries. Single-nucleotide variants are visualized along with protein annotation in a user-friendly web interface. The server offers an effective querying system, which allows to compare variability among different species and accessions, to generate custom data sets based on shared functional features or to perform sequence searches. A documented set of exposed RESTful endpoints make the data accessible programmatically by third-party clients.

Genomic analysis of Sparus aurata reveals the evolutionary dynamics of sex-biased genes in a sequential hermaphrodite fish.

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of Sparus aurata, a sequential hermaphrodite fish. Evolutionary comparative analysis reveals that sex-biased genes in S. aurata are similar in number and function, but evolved following strikingly divergent patterns compared with gonochoristic species, showing overall slower rates because of stronger functional constraints. Fast evolution is observed only for highly ovary-biased genes due to female-specific patterns of selection that are related to the peculiar reproduction mode of S. aurata, first maturing as male, then as female. To our knowledge, these findings represent the first genome-wide analysis on sex-biased loci in a hermaphrodite vertebrate species, demonstrating how having two sexes in the same individual profoundly affects the fate of a large set of evolutionarily relevant genes.

Unique resistance traits against downy mildew from the center of origin of grapevine (Vitis vinifera).

The Eurasian grapevine (Vitis vinifera), an Old World species now cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. The cultivation of resistant V. vinifera varieties would be a sustainable way to reduce the damage caused by the pathogen and the impact of disease management, which involves the economic, health and environmental costs of frequent fungicide application. We report the finding of unique downy mildew resistance traits in a winemaking cultivar from the domestication center of V. vinifera, and characterize the expression of a range of genes associated with the resistance mechanism. Based on comparative experimental inoculations, confocal microscopy and transcriptomics analyses, our study shows that V. vinifera cv. Mgaloblishvili, native to Georgia (South Caucasus), exhibits unique resistance traits against P. viticola. Its defense response, leading to a limitation of P. viticola growth and sporulation, is determined by the overexpression of genes related to pathogen recognition, the ethylene signaling pathway, synthesis of antimicrobial compounds and enzymes, and the development of structural barriers. The unique resistant traits found in Mgaloblishvili highlight the presence of a rare defense system in V. vinifera against P. viticola which promises fresh opportunities for grapevine genetic improvement.

Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars.

KEY MESSAGE: Data obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads. The reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25 S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S 5 , S 6a , S 6b , S 8 , S 11 , S 23 , S 39 , S 46 , S 50 and S 58 ). Moreover, sequence divergence clearly suggests that alleles S 6a and S 6b , proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles.

The genome sequence and transcriptome of Potentilla micrantha and their comparison to Fragaria vesca (the woodland strawberry).

Background: The genus Potentilla is closely related to that of Fragaria, the economically important strawberry genus. Potentilla micrantha is a species that does not develop berries but shares numerous morphological and ecological characteristics with Fragaria vesca. These similarities make P. micrantha an attractive choice for comparative genomics studies with F. vesca. Findings: In this study, the P. micrantha genome was sequenced and annotated, and RNA-Seq data from the different developmental stages of flowering and fruiting were used to develop a set of gene predictions. A 327 Mbp sequence and annotation of the genome of P. micrantha, spanning 2674 sequence contigs, with an N50 size of 335,712, estimated to cover 80% of the total genome size of the species was developed. The genus Potentilla has a characteristically larger genome size than Fragaria, but the recovered sequence scaffolds were remarkably collinear at the micro-syntenic level with the genome of F. vesca, its closest sequenced relative. A total of 33,602 genes were predicted, and 95.1% of bench-marking universal single-copy orthologous genes were complete within the presented sequence. Thus, we argue that the majority of the gene-rich regions of the genome have been sequenced. Conclusions: Comparisons of RNA-Seq data from the stages of floral and fruit development revealed genes differentially expressed between P. micrantha and F. vesca.The data presented are a valuable resource for future studies of berry development in Fragaria and the Rosaceae and they also shed light on the evolution of genome size and organization in this family.

Induction of Terpene Biosynthesis in Berries of Microvine Transformed with VvDXS1 Alleles.

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.