Application of new breeding techniques in fruit trees.

Climate change and rapid adaption of invasive pathogens pose a constant pressure on the fruit industry to develop improved varieties. Aiming to accelerate the development of better-adapted cultivars, new breeding techniques have emerged as a promising alternative to meet the demand of a growing global population. Accelerated breeding, cisgenesis, and CRISPR/Cas genome editing hold significant potential for crop trait improvement and have proven to be useful in several plant species. This review focuses on the successful application of these technologies in fruit trees to confer pathogen resistance and tolerance to abiotic stress and improve quality traits. In addition, we review the optimization and diversification of CRISPR/Cas genome editing tools applied to fruit trees, such as multiplexing, CRISPR/Cas-mediated base editing and site-specific recombination systems. Advances in protoplast regeneration and delivery techniques, including the use of nanoparticles and viral-derived replicons, are described for the obtention of exogenous DNA-free fruit tree species. The regulatory landscape and broader social acceptability for cisgenesis and CRISPR/Cas genome editing are also discussed. Altogether, this review provides an overview of the versatility of applications for fruit crop improvement, as well as current challenges that deserve attention for further optimization and potential implementation of new breeding techniques.

Beyond skin-deep: targeting the plant surface for crop improvement.

The above-ground plant surface is a well-adapted tissue layer that acts as an interface between the plant and its surrounding environment. As such, its primary role is to protect against desiccation and maintain the gaseous exchange required for photosynthesis. Further, this surface layer provides a barrier against pathogens and herbivory, while attracting pollinators and agents of seed dispersal. In the context of agriculture, the plant surface is strongly linked to post-harvest crop quality and yield. The epidermal layer contains several unique cell types adapted for these functions, while the non-lignified above-ground plant organs are covered by a hydrophobic cuticular membrane. This review aims to provide an overview of the latest understanding of the molecular mechanisms underlying crop cuticle and epidermal cell formation, with focus placed on genetic elements contributing towards quality, yield, drought tolerance, herbivory defence, pathogen resistance, pollinator attraction, and sterility, while highlighting the inter-relatedness of plant surface development and traits. Potential crop improvement strategies utilizing this knowledge are outlined in the context of the recent development of new breeding techniques.

Grapevine genome analysis demonstrates the role of gene copy number variation in the formation of monoterpenes.

Volatile organic compounds such as terpenes influence the quality parameters of grapevine through their contribution to the flavour and aroma profile of berries. Biosynthesis of volatile organic compounds in grapevine is relatively complex and controlled by multiple genes, the majority of which are unknown or uncharacterised. To identify the genomic regions that associate with modulation of these compounds in grapevine berries, volatile metabolic data generated via GC-MS from a grapevine mapping population was used to identify quantitative trait loci (QTLs). Several significant QTLs were associated with terpenes, and candidate genes were proposed for sesquiterpene and monoterpene biosynthesis. For monoterpenes, loci on chromosomes 12 and 13 were shown to be associated with geraniol and cyclic monoterpene accumulation, respectively. The locus on chromosome 12 was shown to contain a geraniol synthase gene (VvGer), while the locus on chromosome 13 contained an α-terpineol synthase gene (VvTer). Molecular and genomic investigation of VvGer and VvTer revealed that these genes were found in tandemly duplicated clusters, displaying high levels of hemizygosity. Gene copy number analysis further showed that not only did VvTer and VvGer copy numbers vary within the mapping population, but also across recently sequenced Vitis cultivars. Significantly, VvTer copy number correlated with both VvTer gene expression and cyclic monoterpene accumulation in the mapping population. A hypothesis for a hyper-functional VvTer allele linked to increased gene copy number in the mapping population is presented and can potentially lead to selection of cultivars with modulated terpene profiles. The study highlights the impact of VvTPS gene duplication and copy number variation on terpene accumulation in grapevine.

Grapevine mono- and sesquiterpenes: Genetics, metabolism, and ecophysiology.

Mono- and sesquiterpenes are volatile organic compounds which play crucial roles in human perception of table grape and wine flavour and aroma, and as such their biosynthesis has received significant attention. Here, the biosynthesis of mono- and sesquiterpenes in grapevine is reviewed, with a specific focus on the metabolic pathways which lead to formation of these compounds, and the characterised genetic variation underlying modulation of this metabolism. The bottlenecks for terpene precursor formation in the cytosol and plastid are understood to be the HMG-CoA reductase (HMGR) and 1-deoxy-D-xylylose-5-phosphate synthase (DXS) enzymes, respectively, and lead to the formation of prenyldiphosphate precursors. The functional plasticity of the terpene synthase enzymes which act on the prenyldiphosphate precursors allows for the massive variation in observed terpene product accumulation. This diversity is further enhanced in grapevine by significant duplication of genes coding for structurally diverse terpene synthases. Relatively minor nucleotide variations are sufficient to influence both product and substrate specificity of terpene synthase genes, with these variations impacting cultivar-specific aroma profiles. While the importance of these compounds in terms of grape quality is well documented, they also play several interesting roles in the grapevine's ecophysiological interaction with its environment. Mono- and sesquiterpenes are involved in attraction of pollinators, agents of seed dispersal and herbivores, defence against fungal infection, promotion of mutualistic rhizobacteria interaction, and are elevated in conditions of high light radiation. The ever-increasing grapevine genome sequence data will potentially allow for future breeders and biotechnologists to tailor the aroma profiles of novel grapevine cultivars through exploitation of the significant genetic variation observed in terpene synthase genes.

A high-density integrated map for grapevine based on three mapping populations genotyped by the Vitis18K SNP chip.

KEY MESSAGE: We present a high-density integrated map for grapevine, allowing refinement and improved understanding of the grapevine genome, while demonstrating the applicability of the Vitis18K SNP chip for linkage mapping. The improvement of grapevine through biotechnology requires identification of the molecular bases of target traits by studying marker-trait associations. The Vitis18K SNP chip provides a useful genotyping tool for genome-wide marker analysis. Most linkage maps are based on single mapping populations, but an integrated map can increase marker density and show order conservation. Here we present an integrated map based on three mapping populations. The parents consist of the well-known wine cultivars 'Cabernet Sauvignon', 'Corvina' and 'Rhine Riesling', the lesser-known wine variety 'Deckrot', and a table grape selection, G1-7720. Three high-density population maps with an average inter-locus gap ranging from 0.74 to 0.99 cM were developed. These maps show high correlations (0.9965-0.9971) with the reference assembly, containing only 93 markers with large order discrepancies compared to expected physical positions, of which a third is consistent across multiple populations. Moreover, the genetic data aid the further refinement of the grapevine genome assembly, by anchoring 104 yet unanchored scaffolds. From these population maps, an integrated map was constructed which includes 6697 molecular markers and reduces the inter-locus gap distance to 0.60 cM, resulting in the densest integrated map for grapevine thus far. A small number of discrepancies, mainly of short distance, involve 88 markers that remain conflictual across maps. The integrated map shows similar collinearity to the reference assembly (0.9974) as the single maps. This high-density map increases our understanding of the grapevine genome and provides a useful tool for its further characterization and the dissection of complex traits.

Analysis of wild tomato introgression lines elucidates the genetic basis of transcriptome and metabolome variation underlying fruit traits and pathogen response.

Wild tomato species represent a rich gene pool for numerous desirable traits lost during domestication. Here, we exploited an introgression population representing wild desert-adapted species and a domesticated cultivar to establish the genetic basis of gene expression and chemical variation accompanying the transfer of wild-species-associated fruit traits. Transcriptome and metabolome analysis of 580 lines coupled to pathogen sensitivity assays resulted in the identification of genomic loci associated with levels of hundreds of transcripts and metabolites. These associations occurred in hotspots representing coordinated perturbation of metabolic pathways and ripening-related processes. Here, we identify components of the Solanum alkaloid pathway, as well as genes and metabolites involved in pathogen defense and linking fungal resistance with changes in the fruit ripening regulatory network. Our results outline a framework for understanding metabolism and pathogen resistance during tomato fruit ripening and provide insights into key fruit quality traits.

A Multilevel Study of Melon Fruit Reticulation Provides Insight into Skin Ligno-Suberization Hallmarks.

The skin of fleshy fruit is typically covered by a thick cuticle. Some fruit species develop different forms of layers directly above their skin. Reticulation, for example, is a specialized suberin-based coating that ornaments some commercially important melon (Cucumis melo) fruit and is an important quality trait. Despite its importance, the structural, molecular, and biochemical features associated with reticulation are not fully understood. Here, we performed a multilevel investigation of structural attributes, chemical composition, and gene expression profiles on a set of reticulated and smooth skin melons. High-resolution microscopy, surface profiling, and histochemical staining assays show that reticulation comprises cells with heavily suberized walls accumulating large amounts of typical suberin monomers, as well as lignified cells localized underneath the specialized suberized cell layer. Reticulated skin was characterized by induced expression of biosynthetic genes acting in the core phenylpropanoid, suberin, lignin, and lignan pathways. Transcripts of genes associated with lipid polymer assembly, cell wall organization, and loosening were highly enriched in reticulated skin tissue. These signatures were exclusive to reticulated structures and absent in both the smooth surfaces observed in between reticulated regions and in the skin of smooth fruit. Our data provide important insights into the molecular and metabolic bases of reticulation and its tight association with skin ligno-suberization during melon fruit development. Moreover, these insights are likely to contribute to melon breeding programs aimed at improving postharvest qualities associated with fleshy fruit surface layers.

MYB107 and MYB9 Homologs Regulate Suberin Deposition in Angiosperms.

Suberin, a polymer composed of both aliphatic and aromatic domains, is deposited as a rough matrix upon plant surface damage and during normal growth in the root endodermis, bark, specialized organs (e.g., potato [Solanum tuberosum] tubers), and seed coats. To identify genes associated with the developmental control of suberin deposition, we investigated the chemical composition and transcriptomes of suberized tomato (Solanum lycopersicum) and russet apple (Malus x domestica) fruit surfaces. Consequently, a gene expression signature for suberin polymer assembly was revealed that is highly conserved in angiosperms. Seed permeability assays of knockout mutants corresponding to signature genes revealed regulatory proteins (i.e., AtMYB9 and AtMYB107) required for suberin assembly in the Arabidopsis thaliana seed coat. Seeds of myb107 and myb9 Arabidopsis mutants displayed a significant reduction in suberin monomers and altered levels of other seed coat-associated metabolites. They also exhibited increased permeability, and lower germination capacities under osmotic and salt stress. AtMYB9 and AtMYB107 appear to synchronize the transcriptional induction of aliphatic and aromatic monomer biosynthesis and transport and suberin polymerization in the seed outer integument layer. Collectively, our findings establish a regulatory system controlling developmentally deposited suberin, which likely differs from the one of stress-induced polymer assembly recognized to date.

Solanum pennellii backcross inbred lines (BILs) link small genomic bins with tomato traits.

We present a resource for fine mapping of traits derived from the wild tomato species Solanum pennellii (LA0716). The population of backcross inbred lines (BILs) is composed of 446 lines derived after a few generations of backcrosses of the wild species with cultivated tomato (cultivar M82; LA3475), followed by more than seven generations of self-pollination. The BILs were genotyped using the 10K SOL-CAP single nucleotide polymorphism (SNP) -Chip, and 3700 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs carry, on average, 2.7 introgressions per line, with a mean introgression length of 11.7 Mbp. Whereas the classic 76 introgression lines (ILs) partitioned the genome into 106 mapping bins, the BILs generated 633 bins, thereby enhancing the mapping resolution of traits derived from the wild species. We demonstrate the power of the BILs for rapid fine mapping of simple and complex traits derived from the wild tomato species.

The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly.

The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning.

Genome investigation suggests MdSHN3, an APETALA2-domain transcription factor gene, to be a positive regulator of apple fruit cuticle formation and an inhibitor of russet development.

The outer epidermal layer of apple fruit is covered by a protective cuticle. Composed of a polymerized cutin matrix embedded with waxes, the cuticle is a natural waterproof barrier and protects against several abiotic and biotic stresses. In terms of apple production, the cuticle is essential to maintain long post-harvest storage, while severe failure of the cuticle can result in the formation of a disorder known as russet. Apple russet results from micro-cracking of the cuticle and the formation of a corky suberized layer. This is typically an undesirable consumer trait, and negatively impacts the post-harvest storage of apples. In order to identify genetic factors controlling cuticle biosynthesis (and thus preventing russet) in apple, a quantitative trait locus (QTL) mapping survey was performed on a full-sib population. Two genomic regions located on chromosomes 2 and 15 that could be associated with russeting were identified. Apples with compromised cuticles were identified through a novel and high-throughput tensile analysis of the skin, while histological analysis confirmed cuticle failure in a subset of the progeny. Additional genomic investigation of the determined QTL regions identified a set of underlying genes involved in cuticle biosynthesis. Candidate gene expression profiling by quantitative real-time PCR on a subset of the progeny highlighted the specific expression pattern of a SHN1/WIN1 transcription factor gene (termed MdSHN3) on chromosome 15. Orthologues of SHN1/WIN1 have been previously shown to regulate cuticle formation in Arabidopsis, tomato, and barley. The MdSHN3 transcription factor gene displayed extremely low expression in lines with improper cuticle formation, suggesting it to be a fundamental regulator of cuticle biosynthesis in apple fruit.

Scratching the surface: genetic regulation of cuticle assembly in fleshy fruit.

The hydrophobic cuticular membrane of land plants performs a number of important roles during fruit development, including protection from a range of abiotic and biotic stresses. The components of the fleshy fruit cuticle are synthesized and secreted from the epidermal cells. While the biosynthetic and transport pathways of the cuticle have been thoroughly investigated for a number of decades, the regulatory mechanisms allowing fine tuning of cuticle deposition are only now beginning to be elucidated. Transcription factors belonging to the APETALA2, homeodomain-leucine zipper IV, and MYB families have been shown to be important regulators of both cuticle biosynthesis and epidermal cell differentiation, highlighting the connection between these processes. The involvement of MADS-box transcription factors demonstrates the link between fruit ripening and cuticle deposition. Epigenetic and post-transcriptional regulatory mechanisms also play a role in the control of cuticle biosynthesis, in addition to phytohormones, such as abscisic acid, that have been shown to stimulate cuticle deposition. These various levels of genetic regulation allow the plant constantly to maintain and adjust the cuticle in response to environmental and developmental cues.

Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family.

BACKGROUND: In plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including ß-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-ß-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the "varietal character". Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown. RESULTS: Three grapevine-derived CCD-encoding genes have been isolated and shown to be functional with unique substrate cleavage capacities. Our results demonstrate that the VvCCD4a and VvCCD4b catalyse the cleavage of both linear and cyclic carotenoid substrates. The expression of VvCCD1, VvCCD4a and VvCCD4b was detected in leaf, flower and throughout berry development. VvCCD1 expression was constitutive, whereas VvCCD4a expression was predominant in leaves and VvCCD4b in berries. A transgenic population with a 12-fold range of VvCCD1 expression exhibited a lack of correlation between VvCCD1 expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that the in planta function(s) of VvCCD1 in photosynthetically active tissue is distinct from the in vitro activities demonstrated. The isolation and functional characterisation of VvCCD4a and VvCCD4b identify two additional CCDs that are functional in grapevine. CONCLUSIONS: Taken together, our results indicate that the three CCDs are under various levels of control that include gene expression (spatial and temporal), substrate specificity and compartmentalisation that act individually and/or co-ordinately to maintain carotenoid and volatile apocarotenoid levels in plants. Altering the expression of VvCCD1 in a transgenic grapevine population illustrated the divergence between the in vitro enzyme activity and the in planta activity of this enzyme, thereby contributing to the efforts to understand how enzymatic degradation of carotenoids involved in photosynthesis occurs. The identification and functional characterisation of VvCCD4a and VvCCD4b suggest that these enzymes are primarily responsible for catalysing the cleavage of plastidial carotenoids.

The tomato SlSHINE3 transcription factor regulates fruit cuticle formation and epidermal patterning.

Fleshy tomato fruit typically lacks stomata; therefore, a proper cuticle is particularly vital for fruit development and interaction with the surroundings. Here, we characterized the tomato SlSHINE3 (SlSHN3) transcription factor to extend our limited knowledge regarding the regulation of cuticle formation in fleshy fruits. We created SlSHN3 overexpressing and silenced plants, and used them for detailed analysis of cuticular lipid compositions, phenotypic characterization, and the study on the mode of SlSHN3 action. Heterologous expression of SlSHN3 in Arabidopsis phenocopied overexpression of the Arabidopsis SHNs. Silencing of SlSHN3 results in profound morphological alterations of the fruit epidermis and significant reduction in cuticular lipids. We demonstrated that SlSHN3 activity is mediated by control of genes associated with cutin metabolism and epidermal cell patterning. As with SlSHN3 RNAi lines, mutation in the SlSHN3 target gene, SlCYP86A69, resulted in severe cutin deficiency and altered fruit surface architecture. In vitro activity assays demonstrated that SlCYP86A69 possesses NADPH-dependent ω-hydroxylation activity, particularly of C18:1 fatty acid to the 18-hydroxyoleic acid cutin monomer. This study provided insights into transcriptional mechanisms mediating fleshy fruit cuticle formation and highlighted the link between cutin metabolism and the process of fruit epidermal cell patterning.

The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L.

BACKGROUND: Carotenoids are a heterogeneous group of plant isoprenoids primarily involved in photosynthesis. In plants the cleavage of carotenoids leads to the formation of the phytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in the characteristic flavour and aroma compounds in flowers and fruits and are of specific importance in the varietal character of grapes and wine. This work extends the previous reports of carotenoid gene expression and photosynthetic pigment analysis by providing an up-to-date pathway analysis and an important framework for the analysis of carotenoid metabolic pathways in grapevine. RESULTS: Comparative genomics was used to identify 42 genes putatively involved in carotenoid biosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomes and have been localised to the physical map of the heterozygous ENTAV115 grapevine sequence. Nine of the genes occur as single copies whereas the rest of the carotenoid metabolic genes have more than one paralogue. The cDNA copies of eleven corresponding genes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to be functional. Microarrays provided expression profiles of 39 accessions in the metabolic pathway during three berry developmental stages in Sauvignon blanc, whereas an optimised HPLC analysis provided the concentrations of individual carotenoids. This provides evidence of the functioning of the lutein epoxide cycle and the respective genes in grapevine. Similarly, orthologues of genes leading to the formation of strigolactone involved in shoot branching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoforms typically have different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the three VvNCEDs: Our results support previous findings that VvNCED3 is likely the isoform linked to ABA content in berries. CONCLUSIONS: The carotenoid metabolic pathway is well characterised, and the genes and enzymes have been studied in a number of plants. The study of the 42 carotenoid pathway genes of grapevine showed that they share a high degree of similarity with other eudicots. Expression and pigment profiling of developing berries provided insights into the most complete grapevine carotenoid pathway representation. This study represents an important reference study for further characterisation of carotenoid biosynthesis and catabolism in grapevine.