Diversity of flavour characteristics of table grapes and their contributing volatile compounds analysed by the solvent-assisted flavour evaporation method.

To identify the compounds that contribute to the diverse flavours of table grapes, the flavours and volatile compounds of 38 grape cultivars harvested over 3 years are evaluated through sensory analysis and solvent-assisted flavour evaporation (SAFE). The cultivars are characterized and grouped into seven clusters by hierarchical cluster analysis (HCA) using sensory evaluation data with a flavour wheel specific to table grapes. These clusters were similar to conventional flavour classifications, except that the foxy and neutral cultivars form multiple clusters, highlighting the flavour diversity of table grapes. The SAFE method provides a comprehensive profile of the volatile compounds, including slightly volatile compounds whose profiles are lacking in hybrid grapes and Vitis rotundifolia. The sensory evaluation is supported by the volatile compound profiles, and relationships between the datasets are clarified by multivariate analysis. Specific accumulations and combinations of compounds (α-pinene, ß-pinene, phenylethyl alcohol, furaneol, mesifurane, methyl N-formylanthranilate, and mixed ethyl ester and monoterpenoid) were also identified that contribute to the diversity of flavours (fresh green, floral, fruity, fatty green, sweet, fermented/sour) in table grapes, including linalool and linalool analogues (muscat flavour) along with ethyl ester and hydroxyethyl esters (foxy flavour). The accumulation of these compounds was positively related to a higher flavour intensity. Their specific accumulation and combination supported the flavour diversity of table grapes. This study identified novel flavour-associated compound profiles in table grapes through in-depth volatile compound analysis and non-conventional multivariate analysis.

Ongoing Rapid Evolution of a Post-Y Region Revealed by Chromosome-Scale Genome Assembly of a Hexaploid Monoecious Persimmon (Diospyros kaki).

Plants have evolved sex chromosomes independently in many lineages, and loss of separate sexes can also occur. In this study, we assembled a monoecious recently hexaploidized persimmon (Diospyros kaki), in which the Y chromosome has lost the maleness-determining function. Comparative genomic analysis of D. kaki and its dioecious relatives uncovered the evolutionary process by which the nonfunctional Y chromosome (or Ymonoecy) was derived, which involved silencing of the sex-determining gene, OGI, approximately 2 million years ago. Analyses of the entire X and Ymonoecy chromosomes suggested that D. kaki's nonfunctional male-specific region of the Y chromosome (MSY), which we call a post-MSY, has conserved some characteristics of the original functional MSY. Specifically, comparing the functional MSY in Diospyros lotus and the nonfunctional "post-MSY" in D. kaki indicated that both have been rapidly rearranged, mainly via ongoing transposable element bursts, resembling structural changes often detected in Y-linked regions, some of which can enlarge the nonrecombining regions. The recent evolution of the post-MSY (and possibly also MSYs in dioecious Diospyros species) therefore probably reflects these regions' ancestral location in a pericentromeric region, rather than the presence of male-determining genes and/or genes controlling sexually dimorphic traits.

Genetic basis of lineage-specific evolution of fruit traits in hexaploid persimmon.

Frequent polyploidization events in plants have led to the establishment of many lineage-specific traits representing each species. Little is known about the genetic bases for these specific traits in polyploids, presumably due to plant genomic complexity and their difficulties in applying genetic approaches. Hexaploid Oriental persimmon (Diospyros kaki) has evolved specific fruit characteristics, including wide variations in fruit shapes and astringency. In this study, using whole-genome diploidized/quantitative genotypes from ddRAD-Seq data of 173 persimmon cultivars, we examined their population structures and potential correlations between their structural transitions and variations in nine fruit traits. The population structures of persimmon cultivars were highly randomized and not substantially correlated with the representative fruit traits focused on in this study, except for fruit astringency. With genome-wide association analytic tools considering polyploid alleles, we identified the loci associated with the nine fruit traits; we mainly focused on fruit-shape variations, which have been numerically characterized by principal component analysis of elliptic Fourier descriptors. The genomic regions that putatively underwent selective sweep exhibited no overlap with the loci associated with these persimmon-specific fruit traits. These insights will contribute to understanding the genetic mechanisms by which fruit traits are independently established, possibly due to polyploidization events.

Genome-wide study on the polysomic genetic factors conferring plasticity of flower sexuality in hexaploid persimmon.

Sexuality is one of the fundamental mechanisms that work towards maintaining genetic diversity within a species. In diploid persimmons (Diospyros spp.), separated sexuality, the presence of separate male and female individuals (dioecy), is controlled by the Y chromosome-encoded small-RNA gene, OGI. On the other hand, sexuality in hexaploid Oriental persimmon (Diospyros kaki) is more plastic, with OGI-bearing genetically male individuals, able to produce both male and female flowers (monoecy). This is thought to be linked to the partial inactivation of OGI by a retrotransposon insertion, resulting in DNA methylation of the OGI promoter region. To identify the genetic factors regulating branch sexual conversion, genome-wide correlation/association analyses were conducted using ddRAD-Seq data from an F1 segregating population, and using both quantitative and diploidized genotypes, respectively. We found that allelic ratio at the Y-chromosomal region, including OGI, was correlated with male conversion based on quantitative genotypes, suggesting that OGI can be activated in cis in a dosage-dependent manner. Genome-wide association analysis based on diploidized genotypes, normalized for the effect of OGI allele dosage, detected three fundamental loci associated with male conversion. These loci underlie candidate genes, which could potentially act epigenetically for the activation of OGI expression.

Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which ß-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the ß-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.

Evolution of Lineage-Specific Gene Networks Underlying the Considerable Fruit Shape Diversity in Persimmon.

The shapes of plant organs reflect the evolution of each lineage and have been diversified according to lineage-specific adaptations to environment. Research on the molecular pathways responsible for organ shapes has traditionally been focused mainly on leaves or flowers. Thus, little is known about the pathways controlling fruit shapes, despite their diversity in some plant species. In this study, we analyzed oriental persimmon (Diospyros kaki), which exhibits considerable diversity in fruit shapes among cultivars, to elucidate the underlying molecular mechanism using transcriptomic data and quantitative evaluation. First, to filter the candidate genes associated with persimmon fruit shapes, the whole gene expression patterns obtained using mRNA-Seq analysis from 100 individuals, including a segregated population and various cultivars, were assessed to detect correlations with principal component scores for fruit shapes characterized with elliptic Fourier descriptors. Next, a gene co-expression network analysis with weighted gene co-expression network analysis (WGCNA) package revealed that class 1 KNOX family genes and SEEDSTICK function as integrators along with some phytohormone-related genes, to regulate the fruit shape diversity. On the other hand, the OVATE family genes also contribute to fruit shape diversity, of which pathway would be potentially shared with other plant species. Evolutionary aspects suggest that acquisition of a high lineage-specific and variable expression of class 1 KNOX gene, knotted-like homeobox of Arabidopsis thaliana 1 (KNAT1), in young fruit is important for establishing the persimmon-specific mechanism that determines fruit shape diversity.

Characterization of a gene regulatory network underlying astringency loss in persimmon fruit.

MAIN CONCLUSION: Transcriptome analysis of a persimmon population segregating for an astringency trait in fruit suggested central roles for a limited number of transcriptional regulators in the loss of proanthocyanidin accumulation. Persimmon (Diospyros kaki; 2n = 6x = 90) accumulates a large amount of proanthocyanidins (PAs) in its fruit, resulting in an astringent taste. Persimmon cultivars are classified into four types based on the nature of astringency loss and the amount of PAs at maturity. Pollination constant and non-astringent (PCNA)-type cultivars stop accumulating PAs in the early stages of fruit development and their fruit can be consumed when still firm without the need for artificial deastringency treatments. While the PCNA trait has been shown to be conferred by a recessive allele at a single locus (ASTRINGENCY; AST), the exact genetic determinant remains unidentified. Here, we conducted transcriptome analyses to elucidate the regulatory mechanism underlying this trait using developing fruits of an F1 population segregating for the PCNA trait. Comparisons of the transcriptomes of PCNA and non-PCNA individuals and hierarchical clustering revealed that genes related to the flavonoid pathway and to abiotic stress responses involving light stimulation were expressed coordinately with PA accumulation. Furthermore, coexpression network analyses suggested that three putative transcription factors were central to the PA regulatory network and that at least DkMYB4 and/or DkMYC1, which have been reported to form a protein complex with each other for PA regulation, may have a central role in the differential expression of PA biosynthetic pathway genes between PCNA and non-PCNA.

CRISPR/Cas9-mediated targeted mutagenesis in grape.

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape-an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

Identification of QTLs controlling harvest time and fruit skin color in Japanese pear (Pyrus pyrifolia Nakai).

Using an F1 population from a cross between Japanese pear (Pyrus pyrifolia Nakai) cultivars 'Akiakari' and 'Taihaku', we performed quantitative trait locus (QTL) analysis of seven fruit traits (harvest time, fruit skin color, flesh firmness, fruit weight, acid content, total soluble solids content, and preharvest fruit drop). The constructed simple sequence repeat-based genetic linkage map of 'Akiakari' consisted of 208 loci and spanned 799 cM; that of 'Taihaku' consisted of 275 loci and spanned 1039 cM. Out of significant QTLs, two QTLs for harvest time, one for fruit skin color, and one for flesh firmness were stably detected in two successive years. The QTLs for harvest time were located at the bottom of linkage group (LG) Tai3 (nearest marker: BGA35) and at the top of LG Tai15 (nearest markers: PPACS2 and MEST050), in good accordance with results of genome-wide association study. The PPACS2 gene, a member of the ACC synthase gene family, may control harvest time, preharvest fruit drop, and fruit storage potential. One major QTL associated with fruit skin color was identified at the top of LG 8. QTLs identified in this study would be useful for marker-assisted selection in Japanese pear breeding programs.

S-adenosyl-L-methionine induces compaction of nascent peptide chain inside the ribosomal exit tunnel upon translation arrest in the Arabidopsis CGS1 gene.

Expression of the Arabidopsis CGS1 gene, encoding the first committed enzyme of methionine biosynthesis, is feedback-regulated in response to S-adenosyl-L-methionine (AdoMet) at the mRNA level. This regulation is first preceded by temporal arrest of CGS1 translation elongation at the Ser-94 codon. AdoMet is specifically required for this translation arrest, although the mechanism by which AdoMet acts with the CGS1 nascent peptide remained elusive. We report here that the nascent peptide of CGS1 is induced to form a compact conformation within the exit tunnel of the arrested ribosome in an AdoMet-dependent manner. Cysteine residues introduced into CGS1 nascent peptide showed reduced ability to react with polyethyleneglycol maleimide in the presence of AdoMet, consistent with a shift into the ribosomal exit tunnel. Methylation protection and UV cross-link assays of 28 S rRNA revealed that induced compaction of nascent peptide is associated with specific changes in methylation protection and UV cross-link patterns in the exit tunnel wall. A 14-residue stretch of amino acid sequence, termed the MTO1 region, has been shown to act in cis for CGS1 translation arrest and mRNA degradation. This regulation is lost in the presence of mto1 mutations, which cause single amino acid alterations within MTO1. In this study, both the induced peptide compaction and exit tunnel change were found to be disrupted by mto1 mutations. These results suggest that the MTO1 region participates in the AdoMet-induced arrest of CGS1 translation by mediating changes of the nascent peptide and the exit tunnel wall.