Cell-cycle-linked growth reprogramming encodes developmental time into leaf morphogenesis.

How is time encoded into organ growth and morphogenesis? We address this question by investigating heteroblasty, where leaf development and form are modified with progressing plant age. By combining morphometric analyses, fate-mapping through live-imaging, computational analyses, and genetics, we identify age-dependent changes in cell-cycle-associated growth and histogenesis that underpin leaf heteroblasty. We show that in juvenile leaves, cell proliferation competence is rapidly released in a "proliferation burst" coupled with fast growth, whereas in adult leaves, proliferative growth is sustained for longer and at a slower rate. These effects are mediated by the SPL9 transcription factor in response to inputs from both shoot age and individual leaf maturation along the proximodistal axis. SPL9 acts by activating CyclinD3 family genes, which are sufficient to bypass the requirement for SPL9 in the control of leaf shape and in heteroblastic reprogramming of cellular growth. In conclusion, we have identified a mechanism that bridges across cell, tissue, and whole-organism scales by linking cell-cycle-associated growth control to age-dependent changes in organ geometry.

Genealogical Analyses of 3 Cultivated and 1 Wild Specimen of Vitis vinifera from Greece.

Grapevine (Vitis vinifera) has been an important crop with considerable cultural and economic significance for over 2,500 years, and Greece has been an important entry point into Europe for lineages that were domesticated in Western Asia and the Caucasus. However, whole-genome-based investigation of the demographic history of Greek cultivars relative to other European lineages has only started recently. To understand how Greek cultivars relate to Eurasian domesticated and wild populations, we sequenced 3 iconic domesticated strains ('Xinomavro,' 'Agiorgitiko,' 'Mavrotragano') along with 1 wild accession (the vinetree of Pausanias-a historically important wild specimen) and analyzed their genomic diversity together with a large sample of publicly available domesticated and wild strains. We also reconstructed genealogies by leveraging the powerful tsinfer methodology which has not previously been used in this system. We show that cultivated strains from Greece differ genetically from other strains in Europe. Interestingly, all the 3 cultivated Greek strains clustered with cultivated and wild accessions from Transcaucasia, South Asia, and the Levant and are amongst the very few cultivated European strains belonging to this cluster. Furthermore, our results indicate that 'Xinomavro' shares close genealogical proximity with European elite cultivars such as 'Chardonnay,' 'Riesling,' and 'Gamay' but not 'Pinot.' Therefore, the proximity of 'Xinomavro' to Gouais/Heunisch Weiss is confirmed and the utility of ancestral recombination graph reconstruction approaches to study genealogical relationships in crops is highlighted.

Wild and cultivated olive tree genetic diversity in Greece: a diverse resource in danger of erosion.

The genetic relationships between Greek wild olive tree populations and cultivars were investigated. A total of 219 wild genotypes and 67 cultivar genotypes were analyzed by employing 10 SSR markers. Data evidenced that the wild populations exhibited high levels of genetic diversity and exclusively host 40% of the total number of alleles detected. Inbreeding was observed within populations, probably as a consequence of their fragmented spatial distribution. The genetic differentiation between cultivars and wild individuals, as well as within wild populations, was low. Nevertheless, three gene pools of wild trees were detected, corresponding to the geographical areas of Northeastern Greece, Peloponnese-Crete and Epirus. Most cultivars clustered in a separate group, while the rest of them formed a heterogenous group with membership coefficients akin to the three wild olive clusters. Regarding the history of olive cultivation in Greece, bidirectional gene flow was detected between populations of Peloponnese-Crete and the gene pool that composes some of Greece's most important cultivars, such as "Koroneiki" and "Mastoidis", which is inferred as an indication of a minor domestication event in the area. A strategy for the protection of Greek-oriented olive genetic resources is proposed, along with suggestions for the utilization of the genetically diverse wild resources with regard to the introgression of traits of agronomical interest to cultivars.

Pan-European study of genotypes and phenotypes in the Arabidopsis relative Cardamine hirsuta reveals how adaptation, demography, and development shape diversity patterns.

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species.

Boron stimulates fruit formation and reprograms developmental metabolism in sweet cherry.

Boron modulates a wide range of plant developmental processes; however, the regulation of early fruit development by boron remains poorly defined. We report here the physiological, anatomical, metabolic, and transcriptomic impact of pre-flowering boron supply on the sweet cherry fruit set and development (S1-S5 stages). Our findings revealed that endogenous boron content increased in early growth stages (S1 and S2 stages) following preflowering boron exogenous application. Boron treatment resulted in increased fruit set (S1 and S2 stages) and mesocarp cell enlargement (S2 stage). Various sugars (e.g., fructose and glucose), alcohols (e.g., myo-inositol and maltitol), organic acids (e.g., malic acid and citric acid), amino acids (e.g., valine and serine) accumulated in response to boron application during the various developmental stages (S1-S5 stages). Transcriptomic analysis at early growth (S1 and S2 stages) identified boron-responsive genes that are mainly related to secondary metabolism, amino acid metabolism, calcium-binding, ribosome biogenesis, sugar homeostasis and especially to photosynthesis. We found various boron-induced/repressed genes, including those specifically involved in growth. Several heat shock proteins displayed distinct patterns during the initial growth in boron-exposed fruit. Gene analysis also discovered several putative candidate genes like PavPIP5K9, PavWAT1, PavMIOX, PavCAD1, PavPAL1 and PavSNRK2.7, which could facilitate the investigation of the molecular rationale underlying boron function in early fruit growth. Substantial changes in the expression of numerous transcription factors, including PavbHLH25, PavATHB.12L, and PavZAT10.1,.2 were noticed in fruits exposed to boron. The current study provides a baseline of information for understanding the metabolic processes regulated by boron during sweet cherry fruit early growth and fruit development in general.

Whole genome scanning of a Mediterranean basin hotspot collection provides new insights into olive tree biodiversity and biology.

Olive tree (Olea europaea L. subsp. europaea var. europaea) is one of the most important species of the Mediterranean region and one of the most ancient species domesticated. The availability of whole genome assemblies and annotations of olive tree cultivars and oleaster (O. europaea subsp. europaea var. sylvestris) has contributed to a better understanding of genetic and genomic differences between olive tree cultivars. However, compared to other plant species there is still a lack of genomic resources for olive tree populations that span the entire Mediterranean region. In the present study we developed the most complete genomic variation map and the most comprehensive catalog/resource of molecular variation to date for 89 olive tree genotypes originating from the entire Mediterranean basin, revealing the genetic diversity of this commercially significant crop tree and explaining the divergence/similarity among different variants. Additionally, the monumental ancient tree 'Throuba Naxos' was studied to characterize the potential origin or routes of olive tree domestication. Several candidate genes known to be associated with key agronomic traits, including olive oil quality and fruit yield, were uncovered by a selective sweep scan to be under selection pressure on all olive tree chromosomes. To further exploit the genomic and phenotypic resources obtained from the current work, genome-wide association analyses were performed for 23 morphological and two agronomic traits. Significant associations were detected for eight traits that provide valuable candidates for fruit tree breeding and for deeper understanding of olive tree biology.

A wide foodomics approach coupled with metagenomics elucidates the environmental signature of potatoes.

The term "terroir" has been widely employed to link differential geographic phenotypes with sensorial signatures of agricultural food products, influenced by agricultural practices, soil type, and climate. Nowadays, the geographical indications labeling has been developed to safeguard the quality of plant-derived food that is linked to a certain terroir and is generally considered as an indication of superior organoleptic properties. As the dynamics of agroecosystems are highly intricate, consisting of tangled networks of interactions between plants, microorganisms, and the surrounding environment, the recognition of the key molecular components of terroir fingerprinting remains a great challenge to protect both the origin and the safety of food commodities. Furthermore, the contribution of microbiome as a potential driver of the terroir signature has been underestimated. Herein, we present a first comprehensive view of the multi-omic landscape related to transcriptome, proteome, epigenome, and metagenome of the popular Protected Geographical Indication potatoes of Naxos.

Disclosing the molecular basis of salinity priming in olive trees using proteogenomic model discovery.

Plant responses to salinity are becoming increasingly understood, however, salt priming mechanisms remain unclear, especially in perennial fruit trees. Herein, we showed that low-salt pre-exposure primes olive (Olea europaea) plants against high salinity stress. We then performed a proteogenomic study to characterize priming responses in olive roots and leaves. Integration of transcriptomic and proteomic data along with metabolic data revealed robust salinity changes that exhibit distinct or overlapping patterns in olive tissues, among which we focused on sugar regulation. Using the multi-crossed -omics data set, we showed that major differences between primed and nonprimed tissues are mainly associated with hormone signaling and defense-related interactions. We identified multiple genes and proteins, including known and putative regulators, that reported significant proteomic and transcriptomic changes between primed and nonprimed plants. Evidence also supported the notion that protein post-translational modifications, notably phosphorylations, carbonylations and S-nitrosylations, promote salt priming. The proteome and transcriptome abundance atlas uncovered alterations between mRNA and protein quantities within tissues and salinity conditions. Proteogenomic-driven causal model discovery also unveiled key interaction networks involved in salt priming. Data generated in this study are important resources for understanding salt priming in olive tree and facilitating proteogenomic research in plant physiology.

Study of genetic variation and its association with tensile strength among bamboo species through whole genome resequencing.

Moso bamboo (Phyllostachys edulis) is a versatile plant species that is widely used as a construction material by many low-income countries due to the lack of major construction materials such as steel and reinforced concrete. It is also widely used in China. Bamboo is an economically sustainable material that behaves exceptionally in natural disasters such as earthquakes and it can offer viable solutions for contemporary engineering challenges. Despite bamboo's potential in the engineering sector, biological features such as its long generation time, its large genome size, and its polyploidy are constraining factors for genetic and genomic studies that potentially can assist the breeding efforts. This study re-sequenced 8 Phyllostachys species and 18 natural accessions of Ph. edulis, generating a large set of functionally annotated molecular markers (Single Nucleotide Polymorphisms (SNPs) and InDels) providing key genomic resource information. Moreover, all this genomic information was used to carry out a preliminary genome-wide association analysis and several candidate genes were identified to be correlated with a mechanical property that is of high interest to structural engineers: its tensile strength normal to its fibers (i.e., splitting).

Identification of genes and metabolic pathways involved in wounding-induced kiwifruit ripening.

Fruit is constantly challenged by wounding events, inducing accelerated ripening and irreversible metabolic changes. However, cognate mechanisms that regulate this process are little known. To expand our knowledge of ripening metabolism induced by wounding, an artificial-wound global transcriptome investigation combined with metabolite profiling study was conducted in postharvest kiwifruit (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev. 'Hayward'). Wounding treatment promoted fruit ripening, as demonstrated by changes in fruit firmness, ethylene production and respiration activity determined periodically during a ripening period of 8 d at room temperature. Calcium imaging using fluorescent probe Fluo-3 AM revealed spatial dynamics of Ca2+ signaling in the wounding area following 8d ripening. Several sugars including fructose, glucose, and sucrose as well as organic acids such as citric, succinic and galacturonic acid were increased by wounding. Changes of various amino acids in wounded-treated fruit, especially 5-oxoproline and valine along with alternations of soluble alcohols, like myo-inositol were detected. Gene expression analysis of the wounded fruit showed increased expression of genes that are mainly involved in defense response (e.g., AdTLP.1-3, AdPP2C.1-2, AdMALD1), calcium ion binding (e.g., AdCbEFh, AdCLR, AdANX), TCA cycle (e.g., AdMDH.1, AdMDH.2, AdCS), sugars (e.g., AdSUSA.1, AdSPS4, AdABFr), secondary metabolism (e.g., AdPAL.1-3, AdCCR, AdHCT.1-2), lipid processing (e.g., AdGELP.1-4, AdGELP) and pectin degradation (e.g., AdPE.1-2, AdPAE.1-2, AdPG.1-2) as well as in ethylene (AdERF7, AdERF1B, AdACO.1-4) and auxin (AdICE, AdAEFc, AdASII) synthesis and perception. Moreover, genes related to aquaporins, such as AdAQP2, AdAQP4 and AdAQP7 were down-regulated in fruit exposed to wounding. These results demonstrate multiple metabolic points of wounding regulatory control during kiwifruit ripening and provide insights into the molecular basis of wounding-mediated ripening.

The perennial fruit tree proteogenomics atlas: a spatial map of the sweet cherry proteome and transcriptome.

Genome-wide transcriptome analysis provides systems-level insights into plant biology. Due to the limited depth of quantitative proteomics our understanding of gene-protein-complex stoichiometry is largely unknown in plants. Recently, the complexity of the proteome and its cell-/tissue-specific distribution have boosted the research community to the integration of transcriptomics and proteomics landscapes in a proteogenomic approach. Herein, we generated a quantitative proteome and transcriptome abundance atlas of 15 major sweet cherry (Prunus avium L., cv 'Tragana Edessis') tissues represented by 29 247 genes and 7584 proteins. Additionally, 199 984 alternative splicing events, particularly exon skipping and alternative 3' splicing, were identified in 23 383 transcribed regions of the analyzed tissues. Common signatures as well as differences between mRNA and protein quantities, including genes encoding transcription factors and allergens, within and across the different tissues are reported. Using our integrated dataset, we identified key putative regulators of fruit development, notably genes involved in the biosynthesis of anthocyanins and flavonoids. We also provide proteogenomic-based evidence for the involvement of ethylene signaling and pectin degradation in cherry fruit ripening. Moreover, clusters of genes and proteins with similar and different expression and suppression trends across diverse tissues and developmental stages revealed a relatively low RNA abundance-to-protein correlation. The present proteogenomic analysis allows us to identify 17 novel sweet cherry proteins without prior protein-level annotation evidenced in the currently available databases. To facilitate use by the community, we also developed the Sweet Cherry Atlas Database (https://grcherrydb.com/) for viewing and data mining these resources. This work provides new insights into the proteogenomics workflow in plants and a rich knowledge resource for future investigation of gene and protein functions in Prunus species.

Genotype- and tissue-specific metabolic networks and hub genes involved in water-induced distinct sweet cherry fruit cracking phenotypes.

Sweet cherry fruit cracking is a complex physiological disorder that causes significant economic losses. Despite many years of research there is a lack of understanding of the mechanisms involved in cracking. Here, skin and flesh tissue from the cracking susceptible 'Early Bigi' and the cracking tolerant 'Regina' cultivars were sampled prior and just after water dipping treatment to identify water-affected metabolic networks that putatively involved in fruit cracking. Primary metabolites, most strongly those involved in sugars and amino acid metabolism, such as glucose and asparagine, shifted in 'Early Bigi' compared with 'Regina' tissues following water exposure. Comparisons between cultivars, tissues and dipping points identified significant differentially expressed genes. Particularly, genes related to abscisic acid, ethylene biosynthesis, pectin metabolism, expansins and aquaporins were altered in water-exposed tissues. To further characterize the role of these genes in cracking, their single nucleotide variants of the coding regions was studied in another eight sweet cherry cultivars, which differ in their sensitivity to cracking, revealing a strong link mainly between pectin metabolism-related genes and cracking-phenotypes. Integrated metabolomic and transcriptomic profiling uncovered genotypic- and tissue-specific metabolic pathways, including tricarboxylic acid cycle, cell enlargement, lipid and ethanol biosynthesis, and plant defense that putatively are involved in fruit cracking. Based on these results, a model which describes the skin and flesh metabolic reprogramming during water-induced fruit cracking in the susceptible 'Early Bigi' cultivar is presented. Τhis study can help to explore novel candidate genes and metabolic pathways for cracking tolerance in sweet cherry.

Olive Fruit Development and Ripening: Break on through to the "-Omics" Side.

The olive tree (Olea europaea L. subsp. europaea) is the most important perennial crop in the Mediterranean region, producing table olives and oil, both appreciated for their nutraceutical value. Although olive oil quality traits have been extensively studied, much less attention has been paid to olive drupe. Olive drupe ripening is an extremely complex process involving numerous physiological and molecular changes that are unique in this fruit crop species. This review underlines the contribution of "-omics" techniques and of the recent advances in bioinformatics and analytical tools, notably next-generation sequencing and mass spectrometry, for the characterization of the olive ripening syndrome. The usage of high-dimensional datasets, such as transcriptomics, proteomics, and metabolomics, will provide a systematical description of the molecular-specific processes regulating olive fruit development and ripening. However, the incomplete sequence of the O. europaea L. reference genome has largely hampered the utilization of omics tools towards olive drupe research. Due to this disadvantage, the most reported -omics studies on fruit trees concern metabolomics and only a few transcriptomics and proteomics. In this review, up-to-date applications of -omics technologies towards olive drupe biology are addressed, and future perspectives in olive fruit research are highlighted.

Fruit quality trait discovery and metabolic profiling in sweet cherry genebank collection in Greece.

The current study characterizes the physicochemical, sensory and bioactive compound traits of twenty-two sweet cherry accessions, namely breeding lines, landraces and modern cultivars, embodying the majority of Greek germplasm. The evaluated accessions differ in several quality traits including colour parameters and textural properties as well as sensory attributes, such as taste intensity and overall acceptance. Significant differences in primary metabolites, including fructose, glucose, sorbitol, malic acid were recorded among tested accessions. All genotypes were rich in polyphenols, primarily in quercetin-3,4-O-diglucoside, esculetin, rutin and neochlorogenic acid. An anthocyanins-related discrimination among accessions was also obtained based on cyanidin-3-O-rutinoside and peonidin glycosides content. Overall, the cultivars 'Tsolakeika' and 'Bakirtzeika' exhibited the higher consumer acceptance while the cultivars 'Vasiliadi' and 'Tragana Edessis-Naousis' and especially the breeding line 'TxAg33' contained high polyphenol levels. These results represent a valuable resource for future breeding efforts for sweet cherry cultivars with improved nutritional quality traits.

Whole genome re-sequencing of sweet cherry (Prunus avium L.) yields insights into genomic diversity of a fruit species.

Sweet cherries, Prunus avium L. (Rosaceae), are gaining importance due to their perenniallity and nutritional attributes beneficial for human health. Interestingly, sweet cherry cultivars exhibit a wide range of phenotypic diversity in important agronomic traits, such as flowering time and defense reactions against pathogens. In this study, whole-genome resequencing (WGRS) was employed to characterize genetic variation, population structure and allelic variants in a panel of 20 sweet cherry and one wild cherry genotypes, embodying the majority of cultivated Greek germplasm and a representative of a local wild cherry elite phenotype. The 21 genotypes were sequenced in an average depth of coverage of 33.91×. and effective mapping depth, to the genomic reference sequence of 'Satonishiki' cultivar, between 22.21× to 36.62×. Discriminant analysis of principal components (DAPC) with SNPs revealed two clusters of genotypes. There was a rapid linkage disequilibrium decay, as the majority of SNP pairs with r2 in near complete disequilibrium (>0.8) were found at physical distances less than 10 kb. Functional analysis of the variants showed that the genomic ratio of non-synonymous/synonymous (dN/dS) changes was 1.78. The higher dN frequency in the Greek cohort of sweet cherry could be the result of artificial selection pressure imposed by breeding, in combination with the vegetative propagation of domesticated cultivars through grafting. The majority of SNPs with high impact (e.g., stop codon gaining, frameshift), were identified in genes involved in flowering time, dormancy and defense reactions against pathogens, providing promising resources for future breeding programs. Our study has established the foundation for further large scale characterization of sweet cherry germplasm, enabling breeders to incorporate diverse germplasm and allelic variants to fine tune flowering and maturity time and disease resistance in sweet cherry cultivars.

The complex genetic architecture of shoot growth natural variation in Arabidopsis thaliana.

One of the main outcomes of quantitative genetics approaches to natural variation is to reveal the genetic architecture underlying the phenotypic space. Complex genetic architectures are described as including numerous loci (or alleles) with small-effect and/or low-frequency in the populations, interactions with the genetic background, environment or age. Linkage or association mapping strategies will be more or less sensitive to this complexity, so that we still have an unclear picture of its extent. By combining high-throughput phenotyping under two environmental conditions with classical QTL mapping approaches in multiple Arabidopsis thaliana segregating populations as well as advanced near isogenic lines construction and survey, we have attempted to improve our understanding of quantitative phenotypic variation. Integrative traits such as those related to vegetative growth used in this work (highlighting either cumulative growth, growth rate or morphology) all showed complex and dynamic genetic architecture with respect to the segregating population and condition. The more resolutive our mapping approach, the more complexity we uncover, with several instances of QTLs visible in near isogenic lines but not detected with the initial QTL mapping, indicating that our phenotyping accuracy was less limiting than the mapping resolution with respect to the underlying genetic architecture. In an ultimate approach to resolve this complexity, we intensified our phenotyping effort to target specifically a 3Mb-region known to segregate for a major quantitative trait gene, using a series of selected lines recombined every 100kb. We discovered that at least 3 other independent QTLs had remained hidden in this region, some with trait- or condition-specific effects, or opposite allelic effects. If we were to extrapolate the figures obtained on this specific region in this particular cross to the genome- and species-scale, we would predict hundreds of causative loci of detectable phenotypic effect controlling these growth-related phenotypes.

New Strategies and Tools in Quantitative Genetics: How to Go from the Phenotype to the Genotype.

Quantitative genetics has a long history in plants: It has been used to study specific biological processes, identify the factors important for trait evolution, and breed new crop varieties. These classical approaches to quantitative trait locus mapping have naturally improved with technology. In this review, we show how quantitative genetics has evolved recently in plants and how new developments in phenotyping, population generation, sequencing, gene manipulation, and statistics are rejuvenating both the classical linkage mapping approaches (for example, through nested association mapping) as well as the more recently developed genome-wide association studies. These strategies are complementary in most instances, and indeed, one is often used to confirm the results of the other. Despite significant advances, an emerging trend is that the outcome and efficiency of the different approaches depend greatly on the genetic architecture of the trait in the genetic material under study.