High-resolution genome mapping and functional dissection of chlorogenic acid production in Lonicera maackii.

Amur honeysuckle (Lonicera maackii) is a widely used medicinal plant of the Caprifoliaceae family that produces chlorogenic acid. Research on this plant mainly focuses on its ornamental value and medicinal compounds, but a reference genome sequence and molecular resources for accelerated breeding are currently lacking. Herein, nanopore sequencing and high-throughput chromosome conformation capture (Hi-C) allowed a chromosome-level genome assembly of L. maackii (2n = 18). A global view of the gene regulatory network involved in the biosynthesis of chlorogenic acid and the dynamics of fruit coloration in L. maackii was established through metabolite profiling and transcriptome analyses. Moreover, we identified the genes encoding hydroxycinnamoyl-CoA quinate transferase (LmHQT) and hydroxycinnamoyl-CoA shikimic/quinate transferase (LmHCT), which localized to the cytosol and nucleus. Heterologous overexpression of these genes in Nicotiana benthamiana leaves resulted in elevated chlorogenic acid contents. Importantly, HPLC analyses revealed that LmHCT and LmHQTs recombinant proteins modulate the accumulation of chlorogenic acid (CGA) using quinic acid and caffeoyl CoA as substrates, highlighting the importance of LmHQT and LmHCT in CGA biosynthesis. These results confirmed that LmHQTs and LmHCT catalyze the biosynthesis of CGA in vitro. The genomic data presented in this study will offer a valuable resource for the elucidation of CGA biosynthesis and facilitating selective molecular breeding.

Significance of interstitial fibrosis and p16 in papillary thyroid carcinoma.

We enrolled 264 patients with papillary thyroid carcinoma (PTC). We performed immunohistochemical detection of p16 and determined the degree of interstitial fibrosis (IF). The expression of p16 was associated with pathological tumor-node-metastasis (pTNM) stage and age (p < 0.05). The cancer-specific survival (CSS) was longer in p16-negative patients (195.73 vs. 181.78 months, p = 0.007). p16 was significantly related to the degree of IF (r = 0.130, p = 0.035). PTC patients with no or mild fibrosis tended to have a larger tumor (p = 0.045). The degree of fibrosis was related to the proportion of papillary structure components (p = 0.025). Univariate and multivariate survival analyses showed that relapse-free survival (RFS) was longer in patients with moderate/severe IF (p < 0.05). In summary, p16 was correlated with prognosis and IF of PTC. Patients with moderate/severe IF tend to have better prognosis in RFS.

Elevated levels of interleukin-35 and interleukin-37 in adult patients with obstructive sleep apnea.

BACKGROUND: Systemic inflammation has a critical role in the pathogenesis of obstructive sleep apnea (OSA). Interleukin (IL)-35 and IL-37 have been identified as novel immune-modulating cytokines with anti-inflammatory activities in numerous types of inflammatory disease. The present study aimed to examine the serum levels of IL-35 and IL-37 in patients with OSA, and to investigate their associations with the severity of OSA. METHODS: A total of 97 patients, including 67 cases of OSA and 30 age- and gender-matched healthy control subjects, were enrolled in the present study. All subjects were evaluated by overnight polysomnography. Serum IL-35, IL-37, and pro-inflammatory cytokine IL-1ß levels were examined by ELISA. RESULTS: Compared with those in the control subjects, serum IL-35, IL-37, and IL-1ß levels were significantly elevated in patients with mild, moderate, or severe OSA. Furthermore, a severity-dependent increase in serum IL-35 and IL-37 levels was observed in patients with OSA. IL-35 and IL-37 levels were positively correlated with the apnea-hypopnea index (r = 0.742 and 0.578, respectively; both p < 0.001), while they were negatively correlated with the mean oxygen saturation (r = -0.461 and -0.339, respectively; both p < 0.001) and lowest oxyhaemoglobin saturation (r = -0.616 and -0.463, respectively; both p < 0.001) in patients with OSA. In addition, a positive correlation was observed between IL-35 or IL-37 and IL-1ß levels (all p < 0.001). CONCLUSION: The serum levels of IL-35 and IL-37 were significantly increased in patients with OSA and associated with the severity of OSA, implying that IL-35 and IL-37 may have a protective role in OSA by counteracting inflammatory responses.

The Tetracentron genome provides insight into the early evolution of eudicots and the formation of vessel elements.

BACKGROUND: Tetracentron sinense is an endemic and endangered deciduous tree. It belongs to the Trochodendrales, one of four early diverging lineages of eudicots known for having vesselless secondary wood. Sequencing and resequencing of the T. sinense genome will help us understand eudicot evolution, the genetic basis of tracheary element development, and the genetic diversity of this relict species. RESULTS: Here, we report a chromosome-scale assembly of the T. sinense genome. We assemble the 1.07 Gb genome sequence into 24 chromosomes and annotate 32,690 protein-coding genes. Phylogenomic analyses verify that the Trochodendrales and core eudicots are sister lineages and showed that two whole-genome duplications occurred in the Trochodendrales approximately 82 and 59 million years ago. Synteny analyses suggest that the γ event, resulting in paleohexaploidy, may have only happened in core eudicots. Interestingly, we find that vessel elements are present in T. sinense, which has two orthologs of AtVND7, the master regulator of vessel formation. T. sinense also has several key genes regulated by or regulating TsVND7.2 and their regulatory relationship resembles that in Arabidopsis thaliana. Resequencing and population genomics reveals high levels of genetic diversity of T. sinense and identifies four refugia in China. CONCLUSIONS: The T. sinense genome provides a unique reference for inferring the early evolution of eudicots and the mechanisms underlying vessel element formation. Population genomics analysis of T. sinense reveals its genetic diversity and geographic structure with implications for conservation.

Duplication and diversification of lectin receptor-like kinases (LecRLK) genes in soybean.

Lectin receptor-like kinases (LecRLKs) play important roles in plant development and stress responses. Although genome-wide studies of LecRLKs have been performed in several species, a comprehensive analysis including evolutionary, structural and functional analysis has not been carried out in soybean (Glycine max). In this study, we identified 185 putative LecRLK genes in the soybean genome, including 123 G-type, 60 L-type and 2 C-type LecRLK genes. Tandem duplication and segmental duplication appear to be the main mechanisms of gene expansion in the soybean LecRLK (GmLecRLK) gene family. According to our phylogenetic analysis, G-type and L-type GmLecRLK genes can be organized into fourteen and eight subfamilies, respectively. The subfamilies within the G-type GmLecRLKs differ from each other in gene structure and/or protein domains and motifs, which indicates that the subfamilies have diverged. The evolution of L-type GmLecRLKs has been more conservative: most genes retain the same gene structures and nearly the same protein domain and motif architectures. Furthermore, the expression profiles of G-type and L-type GmLecRLK genes show evidence of functional redundancy and divergence within each group. Our results contribute to a better understanding of the evolution and function of soybean LecRLKs and provide a framework for further functional investigation of them.

Origin and diversification of leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes in plants.

BACKGROUND: Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases in plants and play crucial roles in development and stress responses. The evolutionary relationships among LRR-RLK genes have been investigated in flowering plants; however, no comprehensive studies have been performed for these genes in more ancestral groups. The subfamily classification of LRR-RLK genes in plants, the evolutionary history and driving force for the evolution of each LRR-RLK subfamily remain to be understood. RESULTS: We identified 119 LRR-RLK genes in the Physcomitrella patens moss genome, 67 LRR-RLK genes in the Selaginella moellendorffii lycophyte genome, and no LRR-RLK genes in five green algae genomes. Furthermore, these LRR-RLK sequences, along with previously reported LRR-RLK sequences from Arabidopsis thaliana and Oryza sativa, were subjected to evolutionary analyses. Phylogenetic analyses revealed that plant LRR-RLKs belong to 19 subfamilies, eighteen of which were established in early land plants, and one of which evolved in flowering plants. More importantly, we found that the basic structures of LRR-RLK genes for most subfamilies are established in early land plants and conserved within subfamilies and across different plant lineages, but divergent among subfamilies. In addition, most members of the same subfamily had common protein motif compositions, whereas members of different subfamilies showed variations in protein motif compositions. The unique gene structure and protein motif compositions of each subfamily differentiate the subfamily classifications and, more importantly, provide evidence for functional divergence among LRR-RLK subfamilies. Maximum likelihood analyses showed that some sites within four subfamilies were under positive selection. CONCLUSIONS: Much of the diversity of plant LRR-RLK genes was established in early land plants. Positive selection contributed to the evolution of a few LRR-RLK subfamilies.

Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda.

Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17-50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs.

Widespread and Adaptive Alterations in Genome-Wide Gene Expression Associated with Ecological Divergence of Two Oryza Species.

Ecological speciation is a common mechanism by which new species arise. Despite great efforts, the role of gene expression in ecological divergence and speciation is poorly understood. Here, we conducted a genome-wide gene expression investigation of two Oryza species that are evolutionarily young and distinct in ecology and morphology. Using digital gene expression technology and the paired-end RNA sequencing method, we obtained 21,415 expressed genes across three reproduction-related tissues. Of them, approximately 8% (1,717) differed significantly in expression levels between the two species and these differentially expressed genes are randomly distributed across the genome. Moreover, 62% (1,064) of the differentially expressed genes exhibited a signature of directional selection in at least one species. Importantly, the genes with differential expression between species evolved more rapidly at the 5' flanking sequences than the genes without differential expression relative to coding sequences, suggesting that cis-regulatory changes are likely adaptive and play an important role in the ecological divergence of the two species. Finally, we showed evidence of significant differentiation between species in phenotype traits and observed that genes with differential expression were overrepresented with functional terms involving phenotypic and ecological differentiation between the two species, including reproduction- and stress-related characteristics. Our findings demonstrate that ecological speciation is associated with widespread and adaptive alterations in genome-wide gene expression and provide new insights into the importance of regulatory evolution in ecological speciation in plants.

[Effects of farming managements on the global warming potentials of CH4 and N2O from a rice-wheat rotation system based on the analysis of DNDC modeling].

Taking a rice-wheat rotation system in the suburb of Nanjing, Jiangsu Province of East China as test object, this paper studied the fluxes of CH4 and N2O and their annual dynamics under different farming managements in 2010-2011, and the field observation data were applied to validate the process-based model, denitrification-decomposition (DNDC) model, aimed to approach the applicability of the model to this rotation system, and to use this model to simulate the effects of different environmental factors and farming managements on the global warming potentials (GWPs) of CH4 and N2O. The results showed that except in the treatment control and during wheat growth season, the simulated cumulative emissions of CH4 and N2O from the rotation system in all treatments were basically in coincide with the observed data, the relative deviations being from 7. 1% to 26.3%, and thus, the DNDC model could be applied to simulate the GWPs of cumulative emissions of CH4 and N2O as affected by various environmental factors or management practices. The sensitivity test showed that the GWPs of CH4 and N2O varied significantly with the changes of environmental factors such as the mean annual air temperature, soil bulk density, soil organic carbon, soil texture, and soil pH. Farming managements such as N fertilization, straw returning, and duration of mid-season drainage also had significant effects on the GWPs of CH4 and NO20. Therefore, the above-mentioned environmental factors and farming managements should be taken into account to estimate the greenhouse gases emission from the rice-wheat cropping system on site-specific or regional scale.

Phylogeny of the genus Chrysanthemum L.: evidence from single-copy nuclear gene and chloroplast DNA sequences.

Chrysanthemum L. (Asteraceae-Anthemideae) is a genus with rapid speciation. It comprises about 40 species, most of which are distributed in East Asia. Many of these are narrowly distributed and habitat-specific. Considerable variations in morphology and ploidy are found in this genus. Some species have been the subjects of many studies, but the relationships between Chrysanthemum and its allies and the phylogeny of this genus remain poorly understood. In the present study, 32 species/varieties from Chrysanthemum and 11 from the allied genera were analyzed using DNA sequences of the single-copy nuclear CDS gene and seven cpDNA loci (psbA-trnH, trnC-ycf6, ycf6-psbM, trnY-rpoB, rpS4-trnT, trnL-F, and rpL16). The cpDNA and nuclear CDS gene trees both suggest that 1) Chrysanthemum is not a monophyletic taxon, and the affinity between Chrysanthemum and Ajania is so close that these two genera should be incorporated taxonomically; 2) Phaeostigma is more closely related to the Chrysanthemum+Ajania than other generic allies. According to pollen morphology and to the present cpDNA and CDS data, Ajania purpurea is a member of Phaeostigma. Species differentiation in Chrysanthemum appears to be correlated with geographic and environmental conditions. The Chinese Chrysanthemum species can be divided into two groups, the C. zawadskii group and the C. indicum group. The former is distributed in northern China and the latter in southern China. Many polyploid species, such as C. argyrophyllum, may have originated from allopolyploidization involving divergent progenitors. Considering all the evidence from present and previous studies, we conclude that geographic and ecological factors as well as hybridization and polyploidy play important roles in the divergence and speciation of the genus Chrysanthemum.

Adaptive evolution of the chrysanthemyl diphosphate synthase gene involved in irregular monoterpene metabolism.

BACKGROUND: Chrysanthemyl diphosphate synthase (CDS) is a key enzyme in biosynthetic pathways producing pyrethrins and irregular monoterpenes. These compounds are confined to plants of the tribe Anthemideae of the Asteraceae, and play an important role in defending the plants against herbivorous insects. It has been proposed that the CDS genes arose from duplication of the farnesyl diphosphate synthase (FDS) gene and have different function from FDSs. However, the duplication time toward the origin of CDS and the evolutionary force behind the functional divergence of the CDS gene are still unknown. RESULTS: Two duplication events were detected in the evolutionary history of the FDS gene family in the Asteraceae, and the second duplication led to the origin of CDS. CDS occurred after the divergence of the tribe Mutisieae from other tribes of Asteraceae but before the birth of the Anthemideae tribe. After its origin, CDS accumulated four mutations in sites homologous to the substrate-binding and catalysis sites of FDS. Of these, two sites were involved in the binding of the nucleophilic substrate isopentenyl diphosphate in FDS. Maximum likelihood analyses showed that some sites in CDS were under positive selection and were scattered throughout primary sequences, whereas in the three-dimensional structure model they clustered in the large central cavity. CONCLUSION: Positive selection associated with gene duplication played a major role in the evolution of CDS.

[Distribution characteristics of soil profile nitrous oxide concentration in paddy fields with different rice-upland crop rotation systems].

To investigate the dynamic distribution patterns of nitrous oxide (N2O) in the soil profiles in paddy fields with different rice-upland crop rotation systems, a special soil gas collection device was adopted to monitor the dynamics of N2O at the soil depths 7, 15, 30, and 50 cm in the paddy fields under both flooding and drainage conditions. Two rotation systems were installed, i.e., wheat-single rice and oilseed rape-double rice, each with or without nitrogen (N) application. Comparing with the control, N application promoted the N2O production in the soil profiles significantly (P < 0.01), and there existed significant correlations in the N2O concentration among the four soil depths during the whole observation period (P < 0.01). In the growth seasons of winter wheat and oilseed rape under drainage condition and with or without N application, the N2O concentrations at the soil depths 30 cm and 50 cm were significantly higher than those at the soil depths 7 cm and 15 cm; whereas in the early rice growth season under flooding condition and without N application, the N2O concentrations at the soil depth 7 cm and 15 cm were significantly higher than those at the soil depths 30 cm and 50 cm (P < 0.05). No significant differences were observed in the N2O concentrations at the test soil depths among the other rice cropping treatments. The soil N2O concentrations in the treatments without N application peaked in the transitional period from the upland crops cropping to rice planting, while those in the treatments with N application peaked right after the second topdressing N of upland crops. Relatively high soil N2O concentrations were observed at the transitional period from the upland crops cropping to rice planting.