Fragaria mitogenomes evolve rapidly in structure but slowly in sequence and incur frequent multinucleotide mutations mediated by microinversions.

Plant mitochondrial DNA has been described as evolving rapidly in structure but slowly in sequence. However, many of the noncoding portions of plant mitogenomes are not homologous among species, raising questions about the rate and spectrum of mutations in noncoding regions. Recent studies have suggested that the lack of homology in noncoding regions could be due to increased sequence divergence. We compared 30 kb of coding and 200 kb of noncoding DNA from 13 sequenced Fragaria mitogenomes, followed by analysis of the rate of sequence divergence, microinversion events and structural variations. Substitution rates in synonymous sites and nongenic sites are nearly identical, suggesting that the genome-wide point mutation rate is generally consistent. A surprisingly high number of large multinucleotide substitutions were detected in Fragaria mitogenomes, which may have resulted from microinversion events and could affect phylogenetic signal and local rate estimates. Fragaria mitogenomes preferentially accumulate deletions relative to insertions and substantial genomic arrangements, whereas mutation rates could positively associate with these sequence and structural changes among species. Together, these observations suggest that plant mitogenomes exhibit low point mutations genome-wide but exceptionally high structural variations, and our results favour a gain-and-loss model for the rapid loss of homology among plant mitogenomes.

Oleanolic acid derivatives inhibit the Wnt/ß-catenin signaling pathway by promoting the phosphorylation of ß-catenin in human SMMC-7721 cells.

Oleanolic acid, isolated from privet, has shown antitumor effects in several cancers. However, the underlying molecular mechanism associated with these effects is largely unknown. In this study, we explored the effect of oleanolic acid derivatives on the Wnt/ß-catenin signaling pathway in human hepatocellular carcinoma SMMC-7721 cells. The mRNA and protein levels of related genes were determined by real-time quantitative PCR and Western blot, respectively. Treatment of SMMC-7721 cells with oleanolic acid derivatives led to the downregulation of the mRNA and protein levels of ß-catenin, c-myc, and cyclin D1. Treatment with oleanolic acid derivatives decreased the levels of ß-catenin in both the cytoplasm and the nucleus. Moreover, oleanolic acid derivatives promoted the phosphorylation of ß-catenin (Ser33/37/Thr41) in the cytoplasm. Our results suggest that oleanolic acid derivatives inhibit the Wnt/ß-catenin signaling pathway by stimulating the phosphorylation of ß-catenin (Ser33/37/Thr41) in human SMMC-7721 cells.

Rapid evolution of T2/S-RNase genes in Fragaria linked to multiple transitions from self-incompatibility to self-compatibility.

The T2/RNase gene family is widespread in eukaryotes, and particular members of this family play critical roles in the gametophytic self-incompatibility (GSI) system in plants. Wild diploid strawberry (Fragaria) species have diversified their sexual systems via self-incompatible and self-compatible traits, yet how these traits evolved in Fragaria remains elusive. By integrating the published and de novo assembled genomes and the newly generated RNA-seq data, members of the RNase T2 gene family were systematically identified in six Fragaria species, including three self-incompatible species (Fragaria nipponica, Fragaria nubicola, and Fragaria viridis) and three self-compatible species (Fragaria nilgerrensis, Fragaria vesca, and Fragaria iinumae). In total, 115 RNase T2 genes were identified in the six Fragaria genomes and can be classified into three classes (I-III) according to phylogenetic analysis. The identified RNase T2 genes could be divided into 22 homologous gene sets according to amino acid sequence similarity and phylogenetic and syntenic relationships. We found that extensive gene loss and pseudogenization coupled with small-scale duplications mainly accounted for variations in the RNase T2 gene numbers in Fragaria. Multiple copies of homologous genes were mainly generated from tandem and segmental duplication events. Furthermore, we newly identified five S-RNase genes in three self-incompatible Fragaria genomes, including two in F. nipponica, two in F. viridis, and one in F. nubicola, which fit for typical features of a pistil determinant, including highly pistil-specific expression, highly polymorphic proteins and alkaline isoelectric point (pI), while no S-RNase genes were found in all three self-compatible Fragaria species. Surprisingly, these T2/S-RNase genes contain at least one large intron (>10 kb). This study revealed that the rapid evolution of T2/S-RNase genes within the Fragaria genus could be associated with its sexual mode, and repeated evolution of the self-compatible traits in Fragaria was convergent via losses of S-RNase.

Haplotype-resolved genomes of wild octoploid progenitors illuminate genomic diversifications from wild relatives to cultivated strawberry.

Strawberry is an emerging model for studying polyploid genome evolution and rapid domestication of fruit crops. Here we report haplotype-resolved genomes of two wild octoploids (Fragaria chiloensis and Fragaria virginiana), the progenitor species of cultivated strawberry. Substantial variation is identified between species and between haplotypes. We redefine the four subgenomes and track the genetic contributions of diploid species by additional sequencing of the diploid F. nipponica genome. We provide multiple lines of evidence that F. vesca and F. iinumae, rather than other described extant species, are the closest living relatives of these wild and cultivated octoploids. In response to coexistence with quadruplicate gene copies, the octoploid strawberries have experienced subgenome dominance, homoeologous exchanges and coordinated expression of homoeologous genes. However, some homoeologues have substantially altered expression bias after speciation and during domestication. These findings enhance our understanding of the origin, genome evolution and domestication of strawberries.

Induction of apoptosis by an oleanolic acid derivative in SMMC-7721 human hepatocellular carcinoma cells is associated with mitochondrial dysfunction.

The aim of the present study was to investigate the effects of an oleanolic acid derivative, a novel antitumor drug, on the growth of SMMC-7721 human hepatocellular carcinoma cells and the underlying mechanism. An MTT assay was performed to determine the cytotoxicity of the oleanolic acid derivative. Cell membrane integrity was assessed using fluorescence microscopy to assess the uptake of annexin V-FITC/propidium iodide (PI). Western blotting was used to detect the apoptosis-associated proteins B cell lymphoma-2 (Bcl-2), Bax, caspase-9 and caspase-3. A spectrophotometer was used to analyze the intracellular adenosine triphosphate (ATP) expression level. The loss of mitochondrial membrane potential was detected by performing the JC-1 assay. ELISA was used to evaluate the content of cytochrome c (Cyt-C). The oleanolic acid derivative reduced the cell viability of SMMC-7721 cells in a dose- and time-dependent manner. The half maximal inhibitory concentration values of the oleanolic acid derivative in SMMC-7721 cells at 24, 48 and 72 h were 26.80, 11.85, and 6.66 µM, respectively. The antiapoptotic-protein Bcl-2 was downregulated, and the proapoptotic protein Bax was upregulated following treatment with the oleanolic acid derivative for 48 h. The oleanolic acid derivative induced the cleavage of caspase-9 and caspase-3 as well as promoted annexin V-FITC/PI uptake in SMMC-7721 cells. Furthermore, treatment of SMMC-7721 cells with the oleanolic acid derivative induced a reduction of the intracellular ATP expression level, loss of ΔΨm and Cyt-C release from the mitochondria. The oleanolic acid derivative induced apoptosis in SMMC-7721 human cells. Mitochondrial dysfunction was involved in the anticancer effects of this derivative on SMMC-7721 human cells.

Suppression of the Wnt signaling pathway may contribute to the inhibition of proliferation of human hepatocellular carcinoma SMMC-7721 cells by esculetin.

The aims of the present study were to investigate the effects of esculetin on the proliferation of human hepatocellular carcinoma SMMC-7721 cells and to determine the underlying mechanism behind this activity. An MTT assay was used to assess cell proliferation, reverse transcription-quantitative polymerase chain reaction was used to determine the relative mRNA expression levels of ß-catenin, c-Myc and cyclin D1, and western blot analysis was utilized to determine the levels of the associated proteins. Compared with the dimethyl sulfoxide control, esculetin reduced the cell viability of SMMC-7721 and HL-7702 cells in a dose- and time-dependent manner. Treatment of SMMC-7721 cells with esculetin resulted in downregulation of the mRNA and protein levels of ß-catenin, c-Myc and cyclin D1. Esculetin increased the phosphorylation of ß-catenin at Ser33/Ser37/Thr41 and inhibited the proliferation of human hepatoma SMMC-7721 cells by suppressing the Wnt signaling pathway. The results of the present study suggest that esculetin inhibited the Wnt/ß-catenin signaling pathway in SMMC-7721 cells and may have potential as an effective anti-cancer drug, acting to inhibit the Wnt/ß-catenin signaling pathway.