Multi-hormonal analysis and aquaporins regulation reveal new insights on drought tolerance in grapevine.

Disentangling the factors that foster the tolerance to water stress in plants could provide great benefits to crop productions. In a two-year experiment, two new PIWI (fungus resistant) grapevine varieties, namely Merlot Kanthus and Sauvignon Kretos (Vitis hybrids), grown in the field, were subjected to two different water regimes: weekly irrigated (IR) or not irrigated (NIR) for two months during the summer. The two varieties exhibited large differences in terms of performance under water-limiting conditions. In particular, Merlot Kanthus strongly decreased stem water potential (Ψs) under water shortage and Sauvignon Kretos maintained higher Ψs values accompanied by generally high stomatal conductance and net carbon assimilation, regardless of the treatment. We hypothesized differences in the hormonal profile that mediate most of the plant responses to stresses or in the regulation of the aquaporins that control the water transport in the leaves. In general, substantial differences were found in the abundance of different hormonal classes, with Merlot Kanthus reporting higher concentrations of cytokinins while Sauvignon Kretos higher concentrations of auxins, jasmonate and salicylic acid. Interestingly, under water stress conditions ABA modulation appeared similar between the two cultivars, while other hormones were differently modulated between the two varieties. Regarding the expression of aquaporin encoding genes, Merlot Kanthus showed a significant downregulation of VvPIP2;1 and VvTIP2;1 in leaves exposed to water stress. Both genes have probably a role in influencing leaf conductance, and VvTIP2;1 has been correlated with stomatal conductance values. This evidence suggests that the two PIWI varieties are characterized by different behaviour in response to drought. Furthermore, the findings of the study may be generalized, suggesting the involvement of a complex hormonal cross-talk and aquaporins in effectively influencing plant performance under water shortage.

Boosting grapevine breeding for climate-smart viticulture: from genetic resources to predictive genomics.

The multifaceted nature of climate change is increasing the urgency to select resilient grapevine varieties, or generate new, fitter cultivars, to withstand a multitude of new challenging conditions. The attainment of this goal is hindered by the limiting pace of traditional breeding approaches, which require decades to result in new selections. On the other hand, marker-assisted breeding has proved useful when it comes to traits governed by one or few genes with great effects on the phenotype, but its efficacy is still restricted for complex traits controlled by many loci. On these premises, innovative strategies are emerging which could help guide selection, taking advantage of the genetic diversity within the Vitis genus in its entirety. Multiple germplasm collections are also available as a source of genetic material for the introgression of alleles of interest via adapted and pioneering transformation protocols, which present themselves as promising tools for future applications on a notably recalcitrant species such as grapevine. Genome editing intersects both these strategies, not only by being an alternative to obtain focused changes in a relatively rapid way, but also by supporting a fine-tuning of new genotypes developed with other methods. A review on the state of the art concerning the available genetic resources and the possibilities of use of innovative techniques in aid of selection is presented here to support the production of climate-smart grapevine genotypes.

Juxtaposition of the Source-to-Sink Ratio and Fruit Exposure to Solar Radiation on cv. Merlot (Vitis vinifera L.) Berry Phenolics in a Cool versus Warm Growing Region.

The grapevine source-to-sink ratio and berry exposure to solar radiation both influence grape flavonoid biosynthesis and accumulation. Here, we compared these concepts on cv. Merlot in two different growing locations (Michigan (MI) and Friuli-Venezia Giulia (FVG), IT) to understand whether the environment influences flavonoid sensitivity to these parameters. Three levels of leaf removal (LR0, LR5, LR8) were implemented at the pea-size phenological stage to compare conditions of increased cluster light exposure with a decreasing vine source-to-sink ratio on berry flavonoid accumulation. Treatments did not affect total soluble solids (TSSs) or pH, but titratable acidity (TA) was lower in LR8 at harvest in both locations. LR5 increased anthocyanins and flavonols in MI but decreased most phenolics in FVG. The decreased expression of VviLAR1 and VviF3'5'Hh during ripening supported the lower concentrations of flavan-3-ol monomers and anthocyanins in FVG. In summary, flavonoid biosynthesis and accumulation were more sensitive to solar radiation than the source-to-sink ratio, and the vineyard environment dictated whether solar radiation was beneficial or detrimental to flavonoid biosynthesis.

Insight into Carbohydrate Metabolism and Signaling in Grapevine Buds during Dormancy Progression.

Perennial fruit crops enter dormancy to ensure bud tissue survival during winter. However, a faster phenological advancement caused by global warming exposes bud tissue to a higher risk of spring frost damage. Tissue dehydration and soluble sugars accumulation are connected to freezing tolerance, but non-structural carbohydrates also act as metabolic substrates and signaling molecules. A deepened understanding of sugar metabolism in the context of winter freezing resistance is required to gain insight into adaptive possibilities to cope with climate changes. In this study, the soluble sugar content was measured in a cold-tolerant grapevine hybrid throughout the winter season. Moreover, the expression of drought-responsive hexose transporters VvHT1 and VvHT5, raffinose synthase VvRS and grapevine ABA-, Stress- and Ripening protein VvMSA was analyzed. The general increase in sugars in December and January suggests that they can participate in protecting bud tissues against low temperatures. The modulation of VvHT5, VvINV and VvRS appeared consistent with the availability of the different sugar species; challenging results were obtained for VvHT1 and VvMSA, suggesting interesting hypotheses about their role in the sugar-hormone crosstalk. The multifaceted role of sugars on the intricate phenomenon, which is the response of dormant buds to changing temperature, is discussed.

Cold Hardiness Dynamics and Spring Phenology: Climate-Driven Changes and New Molecular Insights Into Grapevine Adaptive Potential.

Climate change has become a topic of increasing significance in viticulture, severely challenged by this issue. Average global temperatures are increasing, but frost events, with a large variability depending on geographical locations, have been predicted to be a potential risk for grapevine cultivation. Grape cold hardiness encompasses both midwinter and spring frost hardiness, whereas the avoidance of spring frost damage due to late budbreak is crucial in cold resilience. Cold hardiness kinetics and budbreak phenology are closely related and affected by bud's dormancy state. On the other hand, budbreak progress is also affected by temperatures during both winter and spring. Genetic control of bud phenology in grapevine is still largely undiscovered, but several studies have recently aimed at identifying the molecular drivers of cold hardiness loss and the mechanisms that control deacclimation and budbreak. A review of these related traits and their variability in different genotypes is proposed, possibly contributing to develop the sustainability of grapevine production as climate-related challenges rise.

Sugar Metabolism in Stone Fruit: Source-Sink Relationships and Environmental and Agronomical Effects.

The partitioning of assimilates in fruits, which are economically important sink organs, is ruled by different physiological processes and affected by both environmental and agronomical factors. The bulk of the water and solutes, required for growth, is imported into fruits and seeds through xylem and phloem. In the stone fruits, five vascular bundles enter the base of the fruit, then dividing to supply either the flesh or the seed. The main sugars accumulated in stone fruits include fructose, glucose, and sucrose, along with other minor saccharides. The mechanisms of phloem loading in these fruit species have not been fully elucidated yet, but the available data hint either an apoplastic or a symplastic type or possibly a combination of both, depending on the species and the sugar considered. Similarly, phloem unloading mechanisms, elucidated for a small number of species, depend on genotype and developmental stage. Remarkably, key enzymes and transporters involved in the main sugars-conversion and transport pathways have received considerable attention. In stone fruit trees, the presence of an elevated number of fruits alters the source-sink balance, with a consequent intensification of competition among them and between vegetative and reproductive growth. The main environmental factors affecting this balance and the agronomical/artificial manipulations of source-sink relationships to achieve adequate fruit production and quality are reviewed.

Non-structural Carbohydrate Metabolism in the Flesh of Stone Fruits of the Genus Prunus (Rosaceae) - A Review.

Non-structural carbohydrates are abundant constituents of the ripe flesh of all stone fruits. The bulk of their content comprises sucrose, glucose, fructose and sorbitol. However, the abundance of each of these carbohydrates in the flesh differs between species, and also with its stage of development. In this article the import, subcellular compartmentation, contents, metabolism and functions of non-structural carbohydrates in the flesh of commercially cultivated stone fruits of the family Rosaceae are reviewed.

Analysis of Non-Structural Carbohydrates and Xylem Anatomy of Leaf Petioles Offers New Insights in the Drought Response of Two Grapevine Cultivars.

In grapevine, the anatomy of xylem conduits and the non-structural carbohydrates (NSCs) content of the associated living parenchyma are expected to influence water transport under water limitation. In fact, both NSC and xylem features play a role in plant recovery from drought stress. We evaluated these traits in petioles of Cabernet Sauvignon (CS) and Syrah (SY) cultivars during water stress (WS) and recovery. In CS, the stress response was associated to NSC consumption, supporting the hypothesis that starch mobilization is related to an increased supply of maltose and sucrose, putatively involved in drought stress responses at the xylem level. In contrast, in SY, the WS-induced increase in the latter soluble NSCs was maintained even 2 days after re-watering, suggesting a different pattern of utilization of NSC resources. Interestingly, the anatomical analysis revealed that conduits are constitutively wider in SY in well-watered (WW) plants, and that water stress led to the production of narrower conduits only in this cultivar.

Increase in seed tannin extractability and oxidation using a freeze-thaw treatment in cool-climate grown red (Vitis vinifera L.) cultivars.

Grape seed maturation involves the gradual oxidation of tannins, decreasing excessive bitterness and astringency in wine. In cool climates, this process is limited by the short growing season, affecting wine quality. A "freeze-thaw" treatment on seeds of red vinifera cultivars at veraison and harvest was used to evaluate the effect of oxidation and extractability on seed phenolic fractions. Freezing increased the extraction of total phenolics and o-diphenols quantified from fractionation (fraction 1, vacuolar tannins; fraction 2, hydrogen bonded tannins; fraction 3, covalently bonded tannins), especially at harvest. Despite this, colorimetry, microscopy, oxidation reactivity index (ORI), and correlations between the color index and fractions indicated that freezing disrupted vacuole integrity, enhancing oxidation in the seed coat. In conclusion, vacuolar tannins (which are the main seed phenolics extracted during fermentation) were highly correlated with seed color change, potentially providing information for winemaking in cool climate regions.

Resistance to Sharka in Apricot: Comparison of Phase-Reconstructed Resistant and Susceptible Haplotypes of 'Lito' Chromosome 1 and Analysis of Candidate Genes.

Sharka, a common disease among most stone fruit crops, is caused by the Plum Pox Virus (PPV). Resistant genotypes have been found in apricot (Prunus armeniaca L.), one of which-the cultivar 'Lito' heterozygous for the resistance-has been used to map a major quantitative trait locus (QTL) on linkage group 1, following a pseudo-test-cross mating design with 231 individuals. In addition, 19 SNP markers were selected from among the hundreds previously developed, which allowed the region to be limited to 236 kb on chromosome 1. A 'Lito' bacterial artificial chromosome (BAC) library was produced, screened with markers of the region, and positive BAC clones were sequenced. Resistant (R) and susceptible (S) haplotypes were assembled independently. To refine the assembly, the whole genome of 'Lito' was sequenced to high coverage (98×) using PacBio technology, enabling the development of a detailed assembly of the region that was able to predict and annotate the genes in the QTL region. The selected cultivar 'Lito' allowed not only to discriminate structural variants between the two haplotypic regions but also to distinguish specific allele expression, contributing towards mining the PPVres locus. In light of these findings, genes previously indicated (i.e., MATHd genes) to have a possible role in PPV resistance were further analyzed, and new candidates were discussed. Although the results are not conclusive, the accurate and independent assembly of R and S haplotypes of 'Lito' is a valuable resource to predict and test alternative transcription and regulation mechanisms underpinning PPV resistance.

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.

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.

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.

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.

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.

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.

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.