Disruption of NADPH homeostasis by total flavonoids from Adinandra nitida Merr. ex Li leaves triggers ROS-dependent p53 activation leading to apoptosis in non-small cell lung cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Adinandra nitida Merr. ex Li leaves serve as a herbal tea and hold a significant role in traditional Chinese medicine, being applied to assist in tumor treatment. Flavonoids present the primary bioactive constituents in Adinandra nitida Merr. ex Li leaves. AIM OF THE STUDY: To explore the potential of total flavonoids from Adinandra nitida Merr. ex Li Leaves (TFAN) in inhibiting non-small cell lung cancer (NSCLC) and further elucidate the underlying mechanisms. MATERIALS AND METHODS: Human NSCLC cell lines and normal lung cell line were employed to assess the impact of TFAN (0-160 µg/mL for 24, 28 and 72 h) on cell proliferation in vitro. Immunofluorescence (IF) staining gauged p53 expression changes in NSCLC cells under TFAN present condition (150 µg/mL for 24 h). In vivo study utilized NSCLC cell derived xenograft tumors in nude mice, administering TFAN orally (200 and 400 mg/kg) for 14 days. Immunohistochemistry assessed Cleaved Caspase 3 expression change in A549 xenograft tumors treated with TFAN (400 mg/kg for 14 days). RNA-seq and KEGG analysis identified gene expression changes and enriched processes in A549 xenograft tumors treated with TFAN. CM-H2DCFDA and metabolomics assessed ROS level and GSH/GSSG pool changes in A549 cells under TFAN present condition. Cell viability assay and IF staining assessed A549 cell proliferation and p53 expression changes under H2O2-induced oxidative stress (0-40 µM for 24 h) and TFAN present conditions. GSEA and N-Acetyl-L-cysteine (NAC) rescue (0-1 µM for 24 h) analyzed the impact of TFAN on GSH de novo synthesis. NADPH/NADP+ pool measurement and NADPH rescue (0-10 µM for 24 h) analyzed the impact of TFAN on GSH salvage synthesis. GC-FID and HPLC-MS were utilized to detect ethanol and ethyl acetate residues, and to characterize the chemical constituents in TFAN, respectively. The total flavonoid content of TFAN was determined using a 330 nm wavelength. RESULTS: TFAN significantly inhibited A549 cells (wild-type p53) but not NCI-H1299 cells (p53-deficient), NCI-H596 cells (p53-mutant) or BEAS-2B in vitro. IF staining validated p53 genotype for the cell lines and revealed an increase in p53 expression in A549 cells after TFAN treatment. In vivo, TFAN selectively inhibited A549 xenograft tumor growth without discernible toxicity, inducing apoptosis evidenced by Cleaved Caspase 3 upregulation. RNA-seq and KEGG analysis suggested ROS biosynthesis was involved in TFAN-induced p53 activation in A549 cells. Elevated ROS level in TFAN-treated A549 cells were observed. Moreover, TFAN sensitized A549 cells to H2O2-induced oxidative stress, with higher p53 expression. Additionally, A549 cells compensated with GSH de novo synthesis under TFAN present condition, confirmed by GSEA and NAC rescue experiment. TFAN disrupted NADPH homeostasis to impair GSH salvage biosynthesis, supported by NADPH/NADP+ change and NADPH rescue experiment. The chemical constituents of TFAN, with acceptable limits for ethanol and ethyl acetate residues and a total flavonoid content of 68.87%, included Catechin, Epicatechin, Quercitroside, Camellianin A, and Apigenin. CONCLUSION: The disruption of NADPH homeostasis by TFAN triggers ROS-dependent p53 activation that leads to apoptotic cell death, ultimately suppressing NSCLC growth. These findings offer potential therapeutic implications of Adinandra nitida Merr. ex Li leaves in combating NSCLC.

The proposed role of MSL-lncRNAs in causing sex lability of female poplars.

Labile sex expression is frequently observed in dioecious plants, but the underlying genetic mechanism remains largely unknown. Sex plasticity is also observed in many Populus species. Here we carried out a systematic study on a maleness-promoting gene, MSL, detected in the Populus deltoides genome. Our results showed that both strands of MSL contained multiple cis-activating elements, which generated long non-coding RNAs (lncRNAs) promoting maleness. Although female P. deltoides did not have the male-specific MSL gene, a large number of partial sequences with high sequence similarity to this gene were detected in the female poplar genome. Based on sequence alignment, the MSL sequence could be divided into three partial sequences, and heterologous expression of these partial sequences in Arabidopsis confirmed that they could promote maleness. Since activation of the MSL sequences can only result in female sex lability, we propose that MSL-lncRNAs might play a role in causing sex lability of female poplars.

Time-sequential detection of quantitative trait loci and candidate genes underlying the dynamic growth of Salix suchowensis.

Elucidating the genetic factors underlying long-term biological processes remains challenging since the relevant genes and their effects may vary across different developmental stages. In this study, we carried out a large-scale field trial of the progeny of an F1 full-sib pedigree of Salix suchowensis and measured plant height and ground diameter periodically over a time course of 240 days. With the obtained data, we characterized plant growth rhythms and performed time-sequential analyses of quantitative trait loci underlying the dynamic growth of the plants. The dynamic mapping of quantitative trait loci revealed that stem height and ground diameter were under the control of four quantitative trait loci, and the effects of these quantitative trait loci varied greatly throughout the growth process, in which two quantitative trait loci were found to exert a pleiotropic effect determining the correlation between stem height and ground diameter. The analysis of candidate genes in the target genetic intervals showed that the pleiotropic effect of the two quantitative trait loci arises from the colocalization of genes with independent effects on stem height and ground diameter. Further examination of the expression patterns of the candidate genes indicated that height and circumference growth involve different activities of leaf and cambium tissues. This study provides unprecedented information to help us understand the dynamic growth of plants and presents an applicable strategy for elucidating the genetic mechanism underlying a long-term biological process by using plant growth as an example.

Aux/IAA and ARF Gene Families in Salix suchowensis: Identification, Evolution, and Dynamic Transcriptome Profiling During the Plant Growth Process.

The phytohormone auxin plays a pivotal role in the regulation of plant growth and development, including vascular differentiation and tree growth. The auxin/indole-3-acetic acid (Aux/IAA) and auxin response transcription factor (ARF) genes are key components of plant auxin signaling. To gain more insight into the regulation and functional features of Aux/IAA and ARF genes during these processes, we identified 38 AUX/IAA and 34 ARF genes in the genome of Salix suchowensis and characterized their gene structures, conserved domains, and encoded amino acid compositions. Phylogenetic analysis of some typical land plants showed that the Aux/IAA and ARF genes of Salicaceae originated from a common ancestor and were significantly amplified by the ancestral eudicot hexaploidization event and the "salicoid" duplication that occurred before the divergence of poplar and willow. By analyzing dynamic transcriptome profiling data, some Aux/IAA and ARF genes were found to be involved in the regulation of plant growth, especially in the initial plant growth process. Additionally, we found that the expression of several miR160/miR167-ARFs was in agreement with canonical miRNA-ARF interactions, suggesting that miRNAs were possibly involved in the regulation of the auxin signaling pathway and the plant growth process. In summary, this study comprehensively analyzed the sequence features, origin, and expansion of Aux/IAA and ARF genes, and the results provide useful information for further studies on the functional involvement of auxin signaling genes in the plant growth process.

Evidences for a role of two Y-specific genes in sex determination in Populus deltoides.

Almost all plants in the genus Populus are dioecious (i.e. trees are either male or female), but it is unknown whether dioecy evolved in a common ancestor or independently in different subgenera. Here, we sequence the small peritelomeric X- and Y-linked regions of P. deltoides chromosome XIX. Two genes are present only in the Y-linked region. One is a duplication of a non-Y-linked, female-specifically expressed response regulator, which produces siRNAs that block this gene's expression, repressing femaleness. The other is an LTR/Gypsy transposable element family member, which generates long non-coding RNAs. Overexpression of this gene in A. thaliana promotes androecium development. We also find both genes in the sex-determining region of P. simonii, a different poplar subgenus, which suggests that they are both stable components of poplar sex-determining systems. By contrast, only the duplicated response regulator gene is present in the sex-linked regions of P. davidiana and P. tremula. Therefore, findings in our study suggest dioecy may have evolved independently in different poplar subgenera.

The chromosome-scale assembly of the willow genome provides insight into Salicaceae genome evolution.

Salix suchowensis is an early-flowering shrub willow that provides a desirable system for studies on the basic biology of woody plants. The current reference genome of S. suchowensis was assembled with 454 sequencing reads. Here, we report a chromosome-scale assembly of S. suchowensis generated by combining PacBio sequencing with Hi-C technologies. The obtained genome assemblies covered a total length of 356 Mb. The contig N50 of these assemblies was 263,908 bp, which was ~65-fold higher than that reported previously. The contiguity and completeness of the genome were significantly improved. By applying Hi-C data, 339.67 Mb (95.29%) of the assembled sequences were allocated to the 19 chromosomes of haploid willow. With the chromosome-scale assembly, we revealed a series of major chromosomal fissions and fusions that explain the genome divergence between the sister genera of Salix and Populus. The more complete and accurate willow reference genome obtained in this study provides a fundamental resource for studying many genetic and genomic characteristics of woody plants.

Identification of Genes Underlying the Resistance to Melampsora larici-populina in an R Gene Supercluster of the Populus deltoides Genome.

Identification of the particular genes in an R genes supercluster underlying resistance to the rust fungus Melampsora larici-populina in poplar genome remains challenging. Based on the de novo assembly of the Populus deltoides genome, all of the detected major genetic loci conferring resistance to M. larici-populina were confined to a 3.5-Mb region on chromosome 19. The transcriptomes of the resistant and susceptible genotypes were sequenced for a timespan from 0 to 168 hours postinoculation. By mapping the differentially expressed genes to the target genomic region, we identified two constitutive expression R genes and one inducible expression R gene that might confer resistance to M. larici-populina. Nucleotide variations were predicted based on the reconstructed haplotypes for each allele of the candidate genes. We also confirmed that salicylic acid was the phytohormone mediating signal transduction pathways, and PR-1 was identified as a key gene inhibiting rust reproduction. Finally, quantitative reverse transcription PCR assay revealed consistent expressions with the RNA-sequencing data for the detected key genes. This study presents an efficient approach for the identification of particular genes underlying phenotype of interest by the combination of genetic mapping, transcriptome profiling, and candidate gene sequences dissection. The identified key genes would be useful for host resistance diagnosis and for molecular breeding of elite poplar cultivars exhibiting resistance to M. larici-populina infection. The detected R genes are also valuable for testing whether the combination of individual R genes can induce durable quantitative resistance.

Corrigendum #2 to "VGSC: A Web-Based Vector Graph Toolkit of Genome Synteny and Collinearity".

[This corrects the article DOI: 10.1155/2016/7823429.].

Uneven selection pressure accelerating divergence of Populus and Salix.

Populus (poplars) and Salix (willows) are sister genera in the Salicaceae family that arise from a common tetraploid ancestor. The karyotypes of these two lineages are distinguished by two major interchromosomal and some minor intrachromosomal rearrangements, but which one is evolutionarily more primitive remains debatable. In this study, we compare the selection pressure acting on the paralogous genes resulting from salicoid duplication (PGRS) within and between the genomes of the two lineages. Purifying selection was determined to act more strongly on the PGRS in willow than on those in poplar, which would cause a faster loss of paralogous duplicates in willow. Therefore, Salix species are supposed to evolve faster than Populus species, which is consistent with the observation that the former are taxonomically and morphologically more diverse than the latter. In these two lineages, different autosomes were found to have been evolving into sex chromosomes. Examining the ω ratio and the PGRS in the sex determination regions in willow and poplar revealed higher convergent selection pressure and a faster loss of PGRS in the sex determination regions of both lineages. At the chromosome level, the sex chromosome in poplar is characterized by the lowest gene density among all chromosome members, while this feature is not observed on the sex chromosome in willow, suggesting that Populus species may inherit the more incipient sex chromosome from their progenitor. Taken together, Salix is supposed to be the nascent lineage arising from the additional round of genome reorganization that distinguishes the karyotypes of the two sister genera. In this study, assessment of ω ratios also detected a list of paralogous genes under unusual selection pressure, which could have special consequences for the adaptive evolution of Salicaceae species. In conclusion, the results of this study provide unique information for better understanding the genetic mechanism accelerating the divergence of these two closely related lineages.

GEsture: an online hand-drawing tool for gene expression pattern search.

Gene expression profiling data provide useful information for the investigation of biological function and process. However, identifying a specific expression pattern from extensive time series gene expression data is not an easy task. Clustering, a popular method, is often used to classify similar expression genes, however, genes with a 'desirable' or 'user-defined' pattern cannot be efficiently detected by clustering methods. To address these limitations, we developed an online tool called GEsture. Users can draw, or graph a curve using a mouse instead of inputting abstract parameters of clustering methods. GEsture explores genes showing similar, opposite and time-delay expression patterns with a gene expression curve as input from time series datasets. We presented three examples that illustrate the capacity of GEsture in gene hunting while following users' requirements. GEsture also provides visualization tools (such as expression pattern figure, heat map and correlation network) to display the searching results. The result outputs may provide useful information for researchers to understand the targets, function and biological processes of the involved genes.

Organellar genome assembly methods and comparative analysis of horticultural plants.

Although organellar genomes (including chloroplast and mitochondrial genomes) are smaller than nuclear genomes in size and gene number, organellar genomes are very important for the investigation of plant evolution and molecular ecology mechanisms. Few studies have focused on the organellar genomes of horticultural plants. Approximately 1193 chloroplast genomes and 199 mitochondrial genomes of land plants are available in the National Center for Biotechnology Information (NCBI), of which only 39 are from horticultural plants. In this paper, we report an innovative and efficient method for high-quality horticultural organellar genome assembly from next-generation sequencing (NGS) data. Sequencing reads were first assembled by Newbler, Amos, and Minimus software with default parameters. The remaining gaps were then filled through BLASTN search and PCR. The complete DNA sequence was corrected based on Illumina sequencing data using BWA (Burrows-Wheeler Alignment tool) software. The advantage of this approach is that there is no need to isolate organellar DNA from total DNA during sample preparation. Using this procedure, the complete mitochondrial and chloroplast genomes of an ornamental plant, Salix suchowensis, and a fruit tree, Ziziphus jujuba, were identified. This study shows that horticultural plants have similar mitochondrial and chloroplast sequence organization to other seed plants. Most horticultural plants demonstrate a slight bias toward A+T rich features in the mitochondrial genome. In addition, a phylogenetic analysis of 39 horticultural plants based on 15 protein-coding genes showed that some mitochondrial genes are horizontally transferred from chloroplast DNA. Our study will provide an important reference for organellar genome assembly in other horticultural plants. Furthermore, phylogenetic analysis of the organellar genomes of horticultural plants could accurately clarify the unanticipated relationships among these plants.

Detecting the Candidate Gender Determinants by Bioinformatic Prediction of miRNAs and Their Targets from Transcriptome Sequences of the Male and Female Flowers in Salix suchowensis.

MicroRNAs (miRNAs) belong to a class of small, noncoding, and endogenous single-stranded RNAs that negatively regulate gene expression at the posttranscriptional level. Potential miRNAs can be identified based on sequence homology since miRNAs are highly conserved in plants. In this study, we aligned the expressed sequence tags derived from flower buds of male and female S. suchowensis to miRNAs in the miRBase, which enable us to identify 34 potential miRNAs from flower buds of the alternate sexes. Among them, 11 were from the female and 23 were from the male. Analyzing sequence complementarity led to identification of 124 and 55 miRNA targets in the male and female flower buds, respectively. By mapping the target genes of the predicted miRNAs to the sequence assemblies of S. suchowensis, a miR156 mediated gene was detected at the gender locus of willow, which was a transcription factor involved in flower development. It is noteworthy that this target is not expressed in male flower, while it is expressed fairly highly in female flower based on the transcriptome data derived from the alternate sexes of willows. This study provides new bioinformatic clue for further exploring the genetic mechanism underlying gender determination in willows.

Assembly and analysis of the complete Salix purpurea L. (Salicaceae) mitochondrial genome sequence.

Plant mitochondrial (mt) genomes possess several complex features, including a variable size, a dynamic genome structure, and complicated patterns of gene loss and gain throughout evolutionary history. Studies of plant mt genomes can, therefore, provide unique insights into organelle evolution. We assembled the complete Salix purpurea L. mt genome by screening genomic sequence reads generated by a Roche-454 pyrosequencing platform. The pseudo-molecule obtained has a typical circular structure 598,970 bp long, with an overall GC content of 55.06%. The S. purpurea mt genome contains 52 genes: 31 protein-coding, 18 tRNAs, and three rRNAs. Eighteen tandem repeats and 404 microsatellites are distributed unevenly throughout the S. purpurea mt genome. A phylogenetic tree of 23 representative terrestrial plants strongly supports S. purpurea inclusion in the Malpighiales clade. Our analysis contributes toward understanding the organization and evolution of organelle genomes in Salicaceae species.

Corrigendum to "VGSC: A Web-Based Vector Graph Toolkit of Genome Synteny and Collinearity".

[This corrects the article DOI: 10.1155/2016/7823429.].

The Whole Genome Assembly and Comparative Genomic Research of Thellungiella parvula (Extremophile Crucifer) Mitochondrion.

The complete nucleotide sequences of the mitochondrial (mt) genome of an extremophile species Thellungiella parvula (T. parvula) have been determined with the lengths of 255,773 bp. T. parvula mt genome is a circular sequence and contains 32 protein-coding genes, 19 tRNA genes, and three ribosomal RNA genes with a 11.5% coding sequence. The base composition of 27.5% A, 27.5% T, 22.7% C, and 22.3% G in descending order shows a slight bias of 55% AT. Fifty-three repeats were identified in the mitochondrial genome of T. parvula, including 24 direct repeats, 28 tandem repeats (TRs), and one palindromic repeat. Furthermore, a total of 199 perfect microsatellites have been mined with a high A/T content (83.1%) through simple sequence repeat (SSR) analysis and they were distributed unevenly within this mitochondrial genome. We also analyzed other plant mitochondrial genomes' evolution in general, providing clues for the understanding of the evolution of organelles genomes in plants. Comparing with other Brassicaceae species, T. parvula is related to Arabidopsis thaliana whose characters of low temperature resistance have been well documented. This study will provide important genetic tools for other Brassicaceae species research and improve yields of economically important plants.

VGSC: A Web-Based Vector Graph Toolkit of Genome Synteny and Collinearity.

BACKGROUND: In order to understand the colocalization of genetic loci amongst species, synteny and collinearity analysis is a frequent task in comparative genomics research. However many analysis software packages are not effective in visualizing results. Problems include lack of graphic visualization, simple representation, or inextensible format of outputs. Moreover, higher throughput sequencing technology requires higher resolution image output. IMPLEMENTATION: To fill this gap, this paper publishes VGSC, the Vector Graph toolkit of genome Synteny and Collinearity, and its online service, to visualize the synteny and collinearity in the common graphical format, including both raster (JPEG, Bitmap, and PNG) and vector graphic (SVG, EPS, and PDF). RESULT: Users can upload sequence alignments from blast and collinearity relationship from the synteny analysis tools. The website can generate the vector or raster graphical results automatically. We also provide a java-based bytecode binary to enable the command-line execution.

The complete mitochondrial genome of Medicago truncatula.

The complete mitochondrial genome of Medicago truncatula (M. truncatula) was reported in this study. The mitochondrial genome (mitogenome) was assembled to 271 618 nt. The mitogenome contains 31 protein-coding genes, three rRNA genes and 16 tRNAs. The overall base composition of the mitogenome in descending order is A: 27.21%, C: 22.61%, G: 22.78% and T: 27.40%, and the G + C content is 45.39%. Additionally, 30 exons and 17 introns were identified in eight genes and nine tandem repeats were identified with the period size from 10 nt to 33 nt. Phylogenetic analysis shows that the M. truncatula genome is evolutionarily closest to that of Lotus japonicas. With the complete mitogenome of M. truncatula, it is beneficial to the further research of mitogenome of seed plants, and especially helpful for elucidating vital activities of legumes.