Propagation path of a flowering cherry (Cerasus × yedoensis) cultivar 'Somei-Yoshino' traced by somatic mutations.

In the long history of human relations with flowering cherry trees in Japan, 'Somei-Yoshino' occupies an exceptional position among a variety of flowering trees: it is a self-incompatible interspecific hybrid but has been enthusiastically planted by grafting throughout Japan, due most likely to its flamboyant appearance upon full bloom. Thus, 'Somei-Yoshino' gives us a rare opportunity to trace and investigate the occurrence and distribution of somatic mutations within a single plant species through analysis of the genomes of the clonally propagated trees grown under a variety of geographical and artificial environments. In the studies presented here, a total of 46 samples of 'Somei-Yoshino' trees were collected and their genomes were analyzed. We identified 684 single nucleotide mutations, of which 71 were present in more than two samples. Clustering analysis of the mutations indicated that the 46 samples were classified into eight groups, four of which included 36 of the 46 samples analyzed. Interestingly, all the four tree samples collected in Ueno Park of Tokyo were members of the four groups mentioned above. Based on comparative analysis of their mutations, one of the four trees growing in Ueno Park was concluded to be the closest to the original ancestor. We propose that somatic mutations may be used as tracers to establish the ancestral relationship amongst clonally propagated individuals.

Cherry Blossom Forecast Based on Transcriptome of Floral Organs Approaching Blooming in the Flowering Cherry (Cerasus × yedoensis) Cultivar 'Somei-Yoshino'.

To gain insights into the genetic mechanisms underlying blooming and petal movement in flowering cherry (Cerasus × yedoensis), we performed time-course RNA-seq analysis of the floral buds and open-flowers of the most popular flowering cherry cultivar, 'Somei-Yoshino.' Independent biological duplicate samples of floral buds and open-flowers were collected from 'Somei-Yoshino' trees grown at three different locations in Japan. RNA-seq reads obtained from floral bud and open-flower samples collected in the current study (in 2019) and in a previous study (in 2017) were aligned against the genome sequence of 'Somei-Yoshino' to quantify gene transcript levels. Clustering analysis of RNA-seq reads revealed dynamic changes in the transcriptome, with genes in seven modules predominantly expressed at specific time points, ranging from 5 weeks before flowering to 2 weeks after flowering. Based on the identified gene modules and Gene Ontology (GO) terms enriched at different floral stages, we speculate that the genetic mechanisms underlying petal movement and flower opening in cherry involve the processes of development, cell wall organization, reproduction, and metabolism, which are executed by genes encoding transcription factors, phytohormones, transporters, and polysaccharide metabolic enzymes. Furthermore, we established a statistical model for cherry bloom forecasting, based on gene expression levels as RNA markers at different time points before flowering.

Epigenetic Flexibility Underlies Somaclonal Sex Conversions in Hexaploid Persimmon.

Epigenetic regulation adds a flexible layer to genetic variations, potentially enabling long-term, but reversible, changes to a trait, while maintaining genetic information. In the hexaploid Oriental persimmon (Diospyros kaki), genetically monoecious cultivars bearing male flowers require the Y-encoded small RNA (smRNA) gene, OGI. This gene represses the expression of its autosomal counterpart gene, MeGI, as part of the canonical male production system. However, a D. kaki cultivar, Saijo, which lacks the OGI gene and originally bears only female flowers, occasionally produces somaclonal mutant male and revertant female (RF) branches. In this study, we investigated the mechanisms underlying these somaclonal sex conversions in persimmon. Specifically, we aimed to unravel how a genetically female tree without the OGI gene can produce male flowers and RF flowers. Applying multi-omics approaches, we revealed that this noncanonical male production system is basically consistent with the canonical system, in which the accumulation of smRNA targeting MeGI and the considerable DNA methylation of MeGI are involved. The epigenetic status of MeGI on CGN and CHG was synchronized to the genome-wide methylation patterns, both in transition to and from the male production system. These results suggest that the somaclonal sex conversions in persimmon are driven by the genome-wide epigenetic regulatory activities. Moreover, flexibility in the epigenetic layers of long-lived plant species (e.g. trees) is important for overcoming genetic robustness.

Phased genome sequence of an interspecific hybrid flowering cherry, 'Somei-Yoshino' (Cerasus × yedoensis).

We report the phased genome sequence of an interspecific hybrid, the flowering cherry 'Somei-Yoshino' (Cerasus × yedoensis). The sequence data were obtained by single-molecule real-time sequencing technology, split into two subsets based on genome information of the two probable ancestors, and assembled to obtain two haplotype phased genome sequences of the interspecific hybrid. The resultant genome assembly consisting of the two haplotype sequences spanned 690.1 Mb with 4,552 contigs and an N50 length of 1.0 Mb. We predicted 95,076 high-confidence genes, including 94.9% of the core eukaryotic genes. Based on a high-density genetic map, we established a pair of eight pseudomolecule sequences, with highly conserved structures between the two haplotype sequences with 2.4 million sequence variants. A whole genome resequencing analysis of flowering cherries suggested that 'Somei-Yoshino' might be derived from a cross between C. spachiana and either C. speciosa or its relatives. A time-course transcriptome analysis of floral buds and flowers suggested comprehensive changes in gene expression in floral bud development towards flowering. These genome and transcriptome data are expected to provide insights into the evolution and cultivation of flowering cherry and the molecular mechanism underlying flowering.

Recombinant expression, purification, and characterization of polyphenol oxidase 2 (VvPPO2) from "Shine Muscat" (Vitis labruscana Bailey × Vitis vinifera L.).

Polyphenol oxidases (PPOs) catalyze browning reactions in various plant organs, therefore controlling the reactions is important for the food industry. PPOs have been assumed to be involved in skin browning of white grape cultivars; however, the molecular mechanism underlying PPO-mediated browning process remains elusive. We have recently identified a new PPO gene named VvPPO2 from "Shine Muscat" (Vitis labruscana Bailey × V. vinifera L.), and have shown that the gene is transcribed at a higher level than the previously identified VvPPO1 in browning, physiologically disordered berry skins at the maturation stage. In this study, we expressed VvPPO2 in Escherichia coli and, using the purified preparation, revealed unique physicochemical characteristics of the enzyme. Our study opens up a way to not only understand the berry skin browning process but also to elucidate the enzymatic maturation process of grape PPOs.

The Arabidopsis Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproduction.

Intracellular Na(+)/H(+) (NHX) antiporters have important roles in cellular pH and Na(+), K(+) homeostasis. The six Arabidopsis thaliana intracellular NHX members are divided into two groups, endosomal (NHX5 and NHX6) and vacuolar (NHX1 to NHX4). Of the vacuolar members, NHX1 has been characterized functionally, but the remaining members have largely unknown roles. Using reverse genetics, we show that, unlike the single knockouts nhx1 or nhx2, the double knockout nhx1 nhx2 had significantly reduced growth, smaller cells, shorter hypocotyls in etiolated seedlings and abnormal stamens in mature flowers. Filaments of nhx1 nhx2 did not elongate and lacked the ability to dehisce and release pollen, resulting in a near lack of silique formation. Pollen viability and germination was not affected. Quantification of vacuolar pH and intravacuolar K(+) concentrations indicated that nhx1 nhx2 vacuoles were more acidic and accumulated only 30% of the wild-type K(+) concentration, highlighting the roles of NHX1 and NHX2 in mediating vacuolar K(+)/H(+) exchange. Growth under added Na(+), but not K(+), partly rescued the flower and growth phenotypes. Our results demonstrate the roles of NHX1 and NHX2 in regulating intravacuolar K(+) and pH, which are essential to cell expansion and flower development.

The Arabidopsis intracellular Na+/H+ antiporters NHX5 and NHX6 are endosome associated and necessary for plant growth and development.

Intracellular Na(+)/H(+) antiporters (NHXs) play important roles in cellular pH and Na(+) and K(+) homeostasis in all eukaryotes. Based on sequence similarity, the six intracellular Arabidopsis thaliana members are divided into two groups. Unlike the vacuolar NHX1-4, NHX5 and NHX6 are believed to be endosomal; however, little data exist to support either their function or localization. Using reverse genetics, we show that whereas single knockouts nhx5 or nhx6 did not differ from the wild type, the double knockout nhx5 nhx6 showed reduced growth, with smaller and fewer cells and increased sensitivity to salinity. Reduced growth of nhx5 nhx6 was due to slowed cell expansion. Transcriptome analysis indicated that nhx5, nhx6, and the wild type had similar gene expression profiles, whereas transcripts related to vesicular trafficking and abiotic stress were enriched in nhx5 nhx6. We show that unlike other intracellular NHX proteins, NHX5 and NHX6 are associated with punctate, motile cytosolic vesicles, sensitive to Brefeldin A, that colocalize to known Golgi and trans-Golgi network markers. We provide data to show that vacuolar trafficking is affected in nhx5 nhx6. Possible involvements of NHX5 and NHX6 in maintaining organelle pH and ion homeostasis with implications in endosomal sorting and cellular stress responses are discussed.