Prunus mume (mei), which was domesticated in China more than 3,000 years ago as ornamental plant and fruit, is one of the first genomes among Prunus subfamilies of Rosaceae been sequenced. Here, we assemble a 280M genome by combining 101-fold next-generation sequencing and optical mapping data. We further anchor 83.9% of scaffolds to eight chromosomes with genetic map constructed by restriction-site-associated DNA sequencing. Combining P. mume genome with available data, we succeed in reconstructing nine ancestral chromosomes of Rosaceae family, as well as depicting chromosome fusion, fission and duplication history in three major subfamilies. We sequence the transcriptome of various tissues and perform genome-wide analysis to reveal the characteristics of P. mume, including its regulation of early blooming in endodormancy, immune response against bacterial infection and biosynthesis of flower scent. The P. mume genome sequence adds to our understanding of Rosaceae evolution and provides important data for improvement of fruit trees.

Natural killer (NK) cells play an important role in innate immunity against virally infected or transformed cells as the first defense line. Granzyme M (GzmM) is an orphan granzyme that is constitutively highly expressed in NK cells and is consistent with NK cell-mediated cytolysis. We recently demonstrated that GzmM induces caspase-dependent apoptosis with DNA fragmentation through direct cleavage of inhibitor of caspaseactivated DNase (ICAD). However, the molecular mechanisms for GzmM-induced apoptosis are unclear. We found GzmM causes mitochondrial swelling and loss of mitochondrial transmembrane potential. Moreover, GzmM initiates reactive oxygen species (ROS) generation and cytochrome c release. Heat shock protein 75 (HSP75, also known as TRAP1) acts as an antagonist of ROS and protects cells from GzmM-mediated apoptosis. GzmM cleaves TRAP1 and abolishes its antagonistic function to ROS, resulting in ROS accumulation. Silencing TRAP1 through RNA interference increases ROS accumulation, whereas TRAP1 overexpression attenuates ROS production. ROS accumulation is in accordance with the release of cytochrome c from mitochondria and enhances GzmM-mediated apoptosis.

High-density genetic map is a valuable tool for fine mapping locus controlling a specific trait especially for perennial woody plants. In this study, we firstly constructed a high-density genetic map of mei (Prunus mume) using SLAF markers, developed by specific locus amplified fragment sequencing (SLAF-seq). The linkage map contains 8,007 markers, with a mean marker distance of 0.195 cM, making it the densest genetic map for the genus Prunus. Though weeping trees are used worldwide as landscape plants, little is known about weeping controlling gene(s) (Pl). To test the utility of the high-density genetic map, we did fine-scale mapping of this important ornamental trait. In total, three statistic methods were performed progressively based on the result of inheritance analysis. Quantitative trait loci (QTL) analysis initially revealed that a locus on linkage group 7 was strongly responsible for weeping trait. Mutmap-like strategy and extreme linkage analysis were then applied to fine map this locus within 1.14 cM. Bioinformatics analysis of the locus identified some candidate genes. The successful localization of weeping trait strongly indicates that the high-density map constructed using SLAF markers is a worthy reference for mapping important traits for woody plants.

Bud dormancy transition is a vital developmental process for perennial plant survival. The process is precisely regulated by diverse endogenous genetic factors and environmental cues, but the mechanisms are not yet fully understood. Prunus mume is an ideal crop for bud dormancy analysis because of its early spring-flowering characteristics and small sequenced genome. Here, we analyzed the transcriptome profiles at the three endodormancy stages and natural flush stage using RNA sequencing combined with phytohormone and sugar content measurements. Significant alterations in hormone contents and carbohydrate metabolism have been observed, and α-amylases, Glucan Hydrolase Family 17 and diphosphate-glycosyltransferase family might play crucial roles in the interactions between hormones and sugars. The following hypothetical model for understanding the molecular mechanism of bud dormancy in Prunus mume is proposed: low temperatures exposure induces the significant up-regulation of eight C-repeat binding factor genes, which directly promotes all six dormancy-associated MADS-box genes, resulting in dormancy establishment. The prolonged cold and/or subsequently increasing temperature then decreases the expression levels of these two gene families, which alleviates the inhibition of FLOWERING LOCUS T and reopens the growth-promoting pathway, resulting in dormancy release and the initiation of the bud break process.

The TGF-β pathway plays a vital role in development and disease and regulates transcription through a complex composed of receptor-regulated Smads (R-Smads) and Smad4. Extensive biochemical and genetic studies argue that the pathway is activated through R-Smad phosphorylation; however, the dynamics of signaling remain largely unexplored. We monitored signaling and transcriptional dynamics and found that although R-Smads stably translocate to the nucleus under continuous pathway stimulation, transcription of direct targets is transient. Surprisingly, Smad4 nuclear localization is confined to short pulses that coincide with transcriptional activity. Upon perturbation, the dynamics of transcription correlate with Smad4 nuclear localization rather than with R-Smad activity. In Xenopus embryos, Smad4 shows stereotyped, uncorrelated bursts of nuclear localization, but activated RSmads are uniform. Thus, R-Smads relay graded information about ligand levels that is integrated with intrinsic temporal control reflected in Smad4 into the active signaling complex.A small number of signaling pathways are used repeatedly throughout metazoan development, and their effects depend upon timing and context (1). Extensive biochemical characterization of these developmental signaling pathways has elucidated the sequence of events leading from ligand binding at the cell surface to regulation of transcription. Proper temporal control of pathway activity is crucial for normal development; however, the dynamic aspects of signaling are difficult to infer from population data and have lagged behind dissection of pathway components (2, 3). The few cases that have been examined have revealed rich dynamics that could not have been predicted from knowledge of the molecular interactions or from bulk measurements of protein modifications or mRNA levels (2-4).The TGF-β pathway is essential for developmental processes including mesoderm specification and dorsal-ventral axis formation and is dysregulated in a variety of cancers. It also is an important model for pathway crosstalk and dynamics, because it has two branches that share several components including receptors and transcription factors (5, 6). Binding of ligands specific to each branch to receptor complexes leads to the phosphorylation of branch-specific transcription factors: TGF-β/activin/nodal ligands induce the phosphorylation of Smad2/3, whereas bone morphogenic proteins (BMPs) activate Smad1/5/8. Phosphorylation of the receptor-activated Smads (R-Smads) from either branch of the pathway results in complex formation with Smad4, nuclear accumulation, and transcriptional activation.The prevailing model is that R-Smads carry pathway information with Smad4 mirroring their activity (7,8). R-Smad phosphorylation is necessary for the nuclear accumulation and transcriptional activity of both the R-Smads and Smad4. Termination of signaling often is presumed to be caused by either degradation or dephosphorylation of activated R-Smads (9, 10), and it further is assumed that the continuous pres...

Prunus mume is an ornamental flower and fruit tree in Rosaceae. We investigated the GRAS gene family to improve the breeding and cultivation of P. mume and other Rosaceae fruit trees. The GRAS gene family encodes transcriptional regulators that have diverse functions in plant growth and development, such as gibberellin and phytochrome A signal transduction, root radial patterning, and axillary meristem formation and gametogenesis in the P. mume genome. Despite the important roles of these genes in plant growth regulation, no findings on the GRAS genes of P. mume have been reported. In this study, we discerned phylogenetic relationships of P. mume GRAS genes, and their locations, structures in the genome and expression levels of different tissues. Out of 46 identified GRAS genes, 45 were located on the 8 P. mume chromosomes. Phylogenetic results showed that these genes could be classified into 11 groups. We found that Group X was P. mume-specific, and three genes of Group IX clustered with the rice-specific gene Os4. We speculated that these genes existed before the divergence of dicotyledons and monocotyledons and were lost in Arabidopsis. Tissue expression analysis indicated that 13 genes showed high expression levels in roots, stems, leaves, flowers and fruits, and were related to plant growth and development. Functional analysis of 24 GRAS genes and an orthologous relationship analysis indicated that many functioned during plant growth and flower and fruit development. Our bioinformatics analysis provides valuable information to improve the economic, agronomic and ecological benefits of P. mume and other Rosaceae fruit trees.

Late embryogenesis abundant (LEA) proteins play important roles in plant desiccation tolerance. In this study, 30 LEA genes were identified from Chinese plum (Prunus mume) through genome-wide analysis. The PmLEA genes are distributed on all Chinese plum chromosomes except chromosome 3. Twelve (40 %) and five PmLEA genes are arranged in tandem and segmental duplications, respectively. The PmLEA genes could be divided into eight groups (LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, PvLEA18, dehydrin and seed maturation protein). Ten gene conversion events were observed and most of them (70 %) were identified in dehydrin group. Most PmLEA genes were highly expressed in flower (22/30) and up-regulated by ABA treatment (19/30).

Granzyme (Gzm)M is constitutively highly expressed in NK cells that may play a critical role in NK cell-mediated cytolysis. However, the function of GzmM has been less defined. Just one report showed GzmM induces a caspase-independent death pathway. In this study, we demonstrate a protein transfection reagent Pro-Ject can efficiently transport GzmM into target cells. GzmM initiates caspase-dependent apoptosis with typical apoptotic nuclear morphology. GzmM induces DNA fragmentation, not DNA nicking. GzmM can directly degrade inhibitor of caspase-activated DNase to release the nuclease activity of caspase-activated DNase for damaging DNA. Furthermore, GzmM cleaves the DNA damage sensor enzyme poly(ADP-ribose) polymerase to prevent cellular DNA repair and force apoptosis.