Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution

AP1/FUL, SEP, AGL6, and FLC subfamily genes play important roles in flower development. The phylogenetic relationships among them, however, have been controversial, which impedes our understanding of the origin and functional divergence of these genes. One possible reason for the controversy may be the problems caused by changes in the exon-intron structure of genes, which, according to recent studies, may generate non-homologous sites and hamper the homology-based sequence alignment. In this study, we first performed exon-by-exon alignments of these and three outgroup subfamilies (SOC1, AG, and STK). Phylogenetic trees reconstructed based on these matrices show improved resolution and better congruence with species phylogeny. In the context of these phylogenies, we traced evolutionary changes of exon-intron structures in each subfamily. We found that structural changes have occurred frequently following gene duplication and speciation events. Notably, exons 7 and 8 (if present) suffered more structural changes than others. With the knowledge of exon-intron structural changes, we generated more reasonable alignments containing all the focal subfamilies. The resulting trees showed that the SEP subfamily is sister to the monophyletic group formed by AP1/FUL and FLC subfamily genes and that the AGL6 subfamily forms a sister group to the three abovementioned subfamilies. Based on this topology, we inferred the evolutionary history of exon-intron structural changes among different subfamilies. Particularly, we found that the eighth exon originated before the divergence of AP1/FUL, FLC, SEP, and AGL6 subfamilies and degenerated in the ancestral FLC-like gene. These results provide new insights into the origin and evolution of the AP1/FUL, FLC, SEP, and AGL6 subfamilies

Identification and analysis of differential miRNA–mRNA interactions in coronary heart disease: an experimental screening approach

ObjectiveThis aim of this study is to screen the differential molecules of kidney deficiency and blood stasis (KDBS) syndrome in coronary heart disease by high-throughput sequencing. In addition, the study aims to verify the alterations in the expression levels of miR-4685-3p and its regulated downstream, namely, C1QC, C4, and C5, using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), and to determine whether the complement and coagulation cascade pathway is the specific pathogenic pathway.MethodsPatients diagnosed with unstable angina pectoris with KDBS syndrome, patients with non-kidney deficiency blood stasis (NKDBS) syndrome, and a Normal group were recruited. The clinical symptoms of each group were further analyzed. Illumina's NextSeq 2000 sequencing platform and FastQC software were used for RNA sequencing and quality control. DESeq software was used for differential gene expression (DGE) analysis. qPCR and ELISA verification were performed on DGE analysis.ResultsThe DGE profiles of 77 miRNA and 331 mRNA were selected. The GO enrichment analysis comprised 43 biological processes, 49 cell components, and 42 molecular functions. The KEGG enrichment results included 40 KEGG pathways. The PCR results showed that, compared with the Normal group, the miR-4685-3p levels decreased in the CHD_KDBS group (P = 0.001), and were found to be lower than those observed in the CHD_NKDBS group. The downstream mRNA C1 regulated by miR-4685-3p showed an increasing trend in the CHD_KDBS group, which was higher than that in the Normal group (P = 0.0019). The mRNA C4 and C5 in the CHD_KDBS group showed an upward trend, but the difference was not statistically significant. ELISA was utilized for the detection of proteins associated with the complement and coagulation cascade pathway. It was found that the expression level of C1 was significantly upregulated in the CHD_KDBS group compared with the Normal group (P < 0.0001), which was seen to be higher than that in the CHD_NKDBS group (P < 0.0001). The expression levels of C4 and C5 in the CHD_KDBS group were significantly lower than the Normal group, and were lower than that in the CHD_NKDBS group (P < 0.0001).ConclusionThe occurrence of CHD_KDBS might be related to the activation of the complement and coagulation cascade pathway, which is demonstrated by the observed decrease in miR-4685-3p and the subsequent upregulation of its downstream C1QC. In addition, the expression levels of complement C4 and C5 were found to be decreased, which provided a research basis for the prevention and treatment of this disease

The reproductive biology of Calligonum L. in relation to ex situ conservation in a botanical garden

In this study, we observed the flowering phenology, breeding system, pollination and seed germination of four species of Calligonum (C. calliphysa, C. rubicundum, C. densum and C. ebinuricum) in the Turpan Eremophytes Botanic Garden, China. Our results showed that the species had overlapping flowering phenologies and were pollinated by similar pollination agents. Their breeding systems were self-compatible, and with signs of outbreeding, but not of hybridization with each other; the main isolation mechanism was post-zygotic isolation and they also had high seed germination rates. Therefore, they are suited to ex situ conservation in the Turpan Eremophytes Botanic Garden, and can supply sufficient seeds for renewal populations and the conservation of germplasm resources. Furthermore, these results provide theoretical support for the construction of a national germplasm resource garden of Calligonum, and for the introduction to the garden of other eremophyteplants and their conservation

Parallel evolution of apetalous lineages within the buttercup family (Ranunculaceae): outward expansion ofAGAMOUS1, rather than disruption ofAPETALA3-3

Complete loss of petals, or becoming apetalous, has occurred independently in many flowering plant lineages. However, the mechanisms underlying the parallel evolution of naturally occurring apetalous lineages remain largely unclear. Here, by sampling representatives of all nine apetalous genera/tribes of the family Ranunculaceae and conducting detailed morphological, expression, molecular evolutionary and functional studies, we investigate the mechanisms underlying parallel petal losses. We found that while non-expression/downregulation of the petal identity geneAPETALA3-3(AP3-3) is tightly associated with complete petal losses, disruptions of theAP3-3orthologs were unlikely to be the real causes for the parallel evolution of apetalous lineages. We also found that, compared with their close petalous relatives, naturally occurring apetalous taxa usually bear slightly larger numbers of stamens, whereas the number of sepals remains largely unchanged, suggestive of petal-to-stamen rather than petal-to-sepal transformations. In addition, in the recently originated apetalous genusEnemion, the petal-to-stamen transformations have likely been caused by the mutations that led to the elevation and outward expansion of the expression of the C-function gene,AGAMOUS1(AG1). Our results not only provide a general picture of parallel petal losses within the Ranunculaceae but also help understand the mechanisms underlying the independent originations of other apetalous lineages

The making of elaborate petals in Nigella through developmental repatterning

Elaborate petals are present in many flowering plants lineages and have greatly promoted the success and evolutionary radiation of these groups. How elaborate petals are made, however, remains largely unclear. Petals of Nigella (Ranunculaceae) have long been recognized as elaborate and can thus be an excellent model for the study of petal elaboration. Here, by conducting detailed morphological, micromorphological, anatomical, developmental and evolutionary studies on the petals of Nigella species, we explored the processes, general patterns and underlying mechanisms of petal elaboration. We found that petals of Nigella are highly complex, and the complexity can be reflected at various levels. We also found that evolutionary elaboration of the Nigella petals is a gradual process, involving not only modifications of pre-existing structures but also de novo origination of new characters. Further investigations indicated that the elaboration and diversification of Nigella petals were accomplished by modifying the ancestral trajectory of petal development, a process known as developmental repatterning. Our results not only provide new insights into the development and evolution of elaborate petals, but also highlight the necessity of conducting multiple-level investigations for understanding the processes, patterns and underlying mechanisms of plant evolution

Flexibility in the structure of spiral flowers and its underlying mechanisms

Spiral flowers usually bear a variable number of organs, suggestive of the flexibility in structure. The mechanisms underlying the flexibility, however, remain unclear. Here we show that in Nigella damascena, a species with spiral flowers, different types of floral organs show different ranges of variation in number. We also show that the total number of organs per flower is largely dependent on the initial size of the floral meristem, whereas the respective numbers of different types of floral organs are determined by the functional domains of corresponding genetic programmes. By conducting extensive expression and functional studies, we further elucidate the genetic programmes that specify the identities of different types of floral organs. Notably, the AGL6-lineage member NdAGL6, rather than the AP1-lineage members NdFL1/2, is an A-function gene, whereas petaloidy of sepals is not controlled by AP3- or PI-lineage members. Moreover, owing to the formation of a regulatory network, some floral organ identity genes also regulate the boundaries between different types of floral organs. On the basis of these results, we propose that the floral organ identity determination programme is highly dynamic and shows considerable flexibility. Transitions from spiral to whorled flowers, therefore, may be explained by evolution of the mechanisms that reduce the flexibility

Epigenetic-Based Biomarkers in the Malignant Transformation of BEAS-2B Cells Induced by Coal Tar Pitch Extract

Background and Objectives: The carcinogenicity of coal tar pitch (CTP) to occupational workers has been confirmed by the International Agency for Research on Cancer, especially for lung cancer. Herein, we explored the dynamic changes of epigenetic modifications in the malignant transformation process of CTP-induced BEAS-2B cells and also provided clues for screening early biomarkers of CTP-associated occupational lung cancer. Material and Methods: BEAS-2B cells treated with 3.0 μg/mL CTP extract (CTPE) were cultured to the 30th passage to set up a malignant transformation model, which was confirmed by platelet clone formation assay and xenograft assay. DNA methylation levels were determined by ultraviolet-high performance liquid chromatography. mRNA levels in cells and protein levels in supernatants were respectively detected by Real-Time PCR and enzyme-linked immunosorbent assay. Results: The number of clones and the ability of tumor formation in nude mice of CTPE-exposed BEAS-2B cells at 30th passage were significantly increased compared to vehicle control. Moreover, genomic DNA methylation level was down-regulated. The mRNA levels of DNMT1, DNMT3a and HDAC1 as well as the expression of DNMT1 protein were up-regulated since the 10th passage. From the 20th passage, the transcriptional levels of DNMT3b, let-7a and the expression of DNMT3a, DNMT3b, and HDAC1 proteins were detected to be higher than vehicle control, while the level of miR-21 increased only at the 30th passage. Conclusion: Data in this study indicated that the changes of epigenetic molecules including DNMT1, DNMT3a, DNMT3b, HDAC1, and let-7a occurred at the early stages of BEAS-2B cell malignant transformation after CTPE exposure, which provided critical information for screening early biomarkers of CTP-associated occupational lung cancer

Chloroplast genomic data provide new and robust insights into the phylogeny and evolution of the Ranunculaceae

The family Ranunculaceae, a member of early-diverging eudicots that is increasingly being used as a model for the study of plant developmental evolution, has been the focus of systematic studies for centuries. Recent studies showed that the family can be divided into 14 tribes, with Glaucideae, Hydrastideae, and Coptideae being the successive basal-most lineages. The relationships among the remaining 11 tribes, however, remain controversial, so that a clear picture of character evolution within the family is still lacking. In this study, by sequencing, assembling and analyzing the chloroplast (cp) genomes of 35 species representing 31 genera of the 14 tribes, we resolved the relationships among the tribes and genera of the Ranunculaceae and clarified several long-standing controversies. We found that many of the characters that were once widely used for taxonomic and systematic considerations were actually results of parallel, convergent or even reversal evolution, suggestive of un-reliability. We also found that the family has likely experienced two waves of radiative evolution, through which most of the extant tribes and genera were generated. Notably, both waves of radiation were correlated with the increase in the temperature of the earth, suggesting that global warming may have been the driving force of the radiation events. Based on these observations, we hypothesize that global warming and the associated decrease in the type and number of animal pollinators may have been the main reason why taxa with highly elaborate petals as well as those without petal were generated during each of the two waves of radiation