Rachele Falchi

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

Three distinct mutational mechanisms acting on a single gene underpin the origin of yellow flesh in peach

Peach flesh color (white or yellow) is among the most popular commercial criteria for peach classification, and has implications for consumer acceptance and fruit nutritional quality. Despite the increasing interest in improving cultivars of both flesh types, little is known about the genetic basis for the carotenoid content diversity in peach. Here we describe the association between genotypes at a locus encoding the carotenoid cleavage dioxygenase 4 (PpCCD4), localized in pseudomolecule 1 of the Prunus persica reference genome sequence, and the flesh color for 37 peach varieties, including two somatic revertants, and three ancestral relatives of peach, providing definitive evidence that this locus is responsible for flesh color phenotype. We show that yellow peach alleles have arisen from various ancestral haplotypes by at least three independent mutational events involving nucleotide substitutions, small insertions and transposable element insertions, and that these mutations, despite being located within the transcribed portion of the gene, also result in marked differences in transcript levels, presumably as a consequence of differential transcript stability involving nonsense-mediated mRNA decay. The PpCCD4 gene provides a unique example of a gene for which humans, in their quest to diversify phenotypic appearance and qualitative characteristics of a fruit, have been able to select and exploit multiple mutations resulting from a variety of mechanisms.

Identification and differential expression dynamics of peach small GTPases encoding genes during fruit development and ripening

The function of monomeric GTPases of the RAS superfamily in fruit development and ripening has been partially characterized. Here the identification of peach (Prunus persica) small GTPases of the RAS superfamily expressed in fruit and the characterization of their expression profiles during fruit development are described. Extensive searches on expressed sequence tag (EST) databases led to the selection of a total of 24 genes from peach encoding proteins with significant similarity to Arabidopsis small GTPases. Sequence similarity analyses and identification of conserved motifs, diagnostic of specific RAS families and subfamilies, enabled bona fide assignment of fourteen PpRAB, seven PpARF/ARL/SAR, two PpROP and one PpRAN GTPases. Transcriptional expression profiles of peach monomeric GTPases, analysed by real-time quantitative reverse transcription-PCR, were obtained for mesocarp samples, collected in two consecutive years. Reproducible patterns of expression could be identified for five peach RAB-encoding genes (PpRABA1-1, PpRABA2, PpRABD2-1, PpRABD2-2, and PpRABC2), two ARFs (PpARFA1-1 and PpARLB1), and two ROPs (PpROP3 and PpROP4). Interestingly, the transient transcriptional up-regulation of PpARF genes and of PpRAB genes of the A and D clades, putatively controlling the exocytic delivery of cell wall components and modifying enzymes, appeared to coincide with peaks of growth speed and sugar accumulation and with the final phases of ripening. To our knowledge, this is the first description of the co-ordinated differential expression of a set of genes encoding small GTPases of the ARF and RAB families which takes place during key moments of fruit development and maturation.

Sucrose transport and phloem unloading in peach fruit: potential role of two transporters localized in different cell types

Several complex physiological processes, which include long-distance translocation in the phloem and unloading in sink tissues, govern the partitioning of sugars in economically important organs, such as peach fruit. In this study, we took advantage of a symplastic tracer, carboxyfluorescein (CF), providing evidence for an apoplastic sucrose transfer in the early (SI) and middle (SIII) phases of peach fruit development. Moreover, using a combination of in situ hybridization and laser microdissection-assisted expression analysis, three putative sucrose transporters encoding genes (PpSUT1, PpSUT2, PpSUT4) were transcriptionally analyzed to relate their expression with sucrose storage in this organ. Our study revealed that PpSUT2 and PpSUT4 are the genes predominantly expressed in fruit flesh, and the detailed analysis of their expression pattern in the different cell types enabled us to suggest a specialized role in sucrose distribution. Both PpSUTs transporters could be involved in the retrieval of sucrose lost from the symplastic continuum of the phloem and, when expressed in parenchyma cells, they could be active in the import of sucrose into sink tissues, via symport from the apoplast. An alternative hypothesis has been proposed and discussed for PpSUT4 because of its putative tonoplastic localization. Taken together, our results provide new insights into the molecular mechanisms underpinning sucrose unloading and accumulation in peach fruit.

Translating time-course gene expression profiles into semi-algebraic hybrid automata via dimensionality reduction

Biotechnological innovations which sample gene expressions allow to measure the gene expression levels of a biological system with varying degree of accuracy, cost and speed. By repeating the measurement steps at different sampling rates, one can both infer relations among the genes and define a dynamic model of the underlying biological system. When a very large number of genes and measurements are involved, they raise several difficult algorithmic questions, as accurate model-building, checking and inference tasks. Semi-algebraic hybrid automata were proposed as a modeling formalism for biological systems (see, e.g., [17,6]), and demonstrated their abilities to handle complex biochemical pathways. This paper proposes an automatic procedure to build semi-algebraic hybrid automata from gene-expression profiles. In order to reduce the size of the resulting automata and to minimize their analysis computational complexity, our approach exploits various dimensionality reduction techniques. The paper concludes with several experimental results about peach fruit.

Expression of peach sucrose transporters in heterologous systems points out their different physiological role

Sucrose is the major phloem-translocated component in a number of economically important plant species. The comprehension of the mechanisms involved in sucrose transport in peach fruit appears particularly relevant, since the accumulation of this sugar, during ripening, is crucial for the growth and quality of the fruit. Here, we report the functional characterisation and subcellular localisation of three sucrose transporters (PpSUT1, PpSUT2, PpSUT4) in peach, and we formulate novel hypotheses about their role in accumulation of sugar. We provide evidence, about the capability of both PpSUT1 and PpSUT4, expressed in mutant yeast strains to transport sucrose. The functionality of PpSUT1 at the plasma membrane, and of PpSUT4 at the tonoplast, has been demonstrated. On the other hand, the functionality of PpSUT2 was not confirmed: this protein is unable to complement two sucrose uptake-deficient mutant yeast strains. Our results corroborate the hypotheses that PpSUT1 partakes in phloem loading in leaves, and PpSUT4 sustains cell metabolism by regulating sucrose efflux from the vacuole.

Leaf Removal and Cluster Thinning Efficiencies Are Highly Modulated by Environmental Conditions in Cool Climate Viticulture

One of several challenges in cool climate viticulture with a short growing season is to consistently reach a uniform, optimal fruit technological maturity at harvest before the first autumn frost. Weather conditions in Michigan from veraison to harvest are highly variable and unpredictable among years, constraining the preharvest assessment of fruit quality for grapegrowers and wineries. Under these environmental conditions, cluster thinning and leaf removal are commonly adopted viticultural techniques to enhance fruit ripening. Cluster thinning consists of a selective elimination of clusters to optimize the source/sink ratio of the vine. Cluster zone leaf removal induces changes in the fruit microenvironment, particularly solar radiation, temperature, and aeration. In this work, we evaluated the effects of cluster thinning and cluster zone leaf removal, applied separately in combination at veraison, on Cabernet franc in two consecutive years, 2011 and 2012. The two seasons had very distinct weather patterns from veraison to harvest. Fruit maturity was enhanced at 15 to 20 days after veraison in both years by these viticultural techniques, but with very different dynamics. The combination of leaf removal and cluster thinning led to greater fruit uniformity and better chemical composition at harvest in 2011, a year characterized by low heat accumulation after veraison. In 2012, when heat accumulation and mean temperatures after veraison were higher than in 2011, no differences were observed among treatments.

Tissue-Specific and Developmental Expression Pattern of Abscisic Acid Biosynthetic Genes in Peach Fruit: Possible Role of the Hormone in the Coordinated Growth of Seed and Mesocarp

Peach fruit tissues start differentiating into mesocarp, endocarp, and seed from the first phases of development. A seed is necessary for fruit set, and it may strongly regulate the growth of the whole fruit through metabolic or hormonal signals. Although the importance of sugar and hormone signaling in growing fruit, such as peach, has been analyzed extensively, no conclusions as to the cross-talk between these signal molecules and their role in seed and flesh development have previously been made. The present study provides insight into the regulatory steps of the carotenoid/ABA biosynthetic pathway to establish possible relationships between growth and accumulation of pigments, sugar, and hormone in the different tissues of peach fruit (Prunus persica L. Batsch, cv Redhaven). In the flesh, the transcriptional pattern of most of the genes involved in the ABA synthesis exhibits a good association with both color changes and hormone accumulation. In contrast, in the seed, along with other nongreen tissues, this association is not conceivable. The behavior of zeaxanthin epoxidase genes (Ppzep) well represents the presence of distinct regulatory mechanisms at various steps of the pathway and in a tissue-specific manner. Moreover, the key role of 9-cis-epoxycarotenoid dioxygenase (NCED) enzyme in regulating ABA synthesis appears to be substantiated by observed Ppnced expression profiles, both in the flesh and in the seed. Based on the results obtained in this study, a crucial connection between ABA biosynthesis, sugar content, and sucrose cleavage enzymes (sucrose synthase), at different stages of fruit development, is proposed.

Combined Effects of Early Season Leaf Removal and Climatic Conditions on Aroma Precursors in Sauvignon Blanc Grapes

Early leaf removal around the cluster zone is a common technique applied in cool climate viticulture, to regulate yield components and improve fruit quality. Despite the increasing amount of information on early leaf removal and its impact on total soluble solids, anthocyanins, and polyphenols, less is known regarding aroma compounds. In order to verify the hypothesis that defoliation, applied before or after flowering, could impact the biosynthesis of thiol precursors, we performed a two year (2013 and 2014) experiment on Sauvignon blanc. We provided evidence that differential accumulation of thiol precursors in berries is affected by the timing of defoliation, and this impact was related to modifications in the biosynthetic pathway. Furthermore, the possible interaction between leaf removal treatment and seasonal weather conditions, and its effect on the biosynthesis of volatile precursors are discussed. Our results suggested that in Sauvignon blanc the relative proportion of 4-S-glutathionyl-4-methylpentan-2-one (G-4MSP) and 3-S-glutathionylhexan-1-ol (G-3SH) precursors can be affected by defoliation, and this could be related to the induction of two specific genes encoding glutathione-S-transferases (VvGST3 and VvGST5), while no significant effects on basic fruit chemical parameters, polyphenols, and methoxypyrazines were ascertained under our experimental conditions.

Enhancement of Fruit Technological Maturity and Alteration of the Flavonoid Metabolomic Profile in Merlot (Vitis vinifera L.) by Early Mechanical Leaf Removal

Removal of basal leaves near blooms inevitably affects grapevine balance and cluster microclimate conditions, improving fruit quality. Mechanization of this practice allows growers to save time and resources, but to our knowledge, it has not yet been compared with the manual application of this practice in a cool-climate region where seasonal temperatures frequently limit fruit technological maturity and phenolic ripening in red Vitis vinifera cultivars. In our research, berry sugar concentration was highest with prebloom mechanical treatment (PB-ME). Furthermore, metabolomics analysis revealed that PB-ME favored the accumulation of significantly more disubstituted anthocyanins and flavonols and OH-substituted anthocyanins compared with manual application. Given that vine balance was similar between treatments, increased ripening with PB-ME is likely due to enhanced microclimate conditions and higher carbon partitioning through a younger canopy containing basal leaf fragments proximal to fruit. This information provides an important strategy for consistently ripening red Vitis vinifera cultivars in cool climates.