Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives.

Despite the economic, ecological, and scientific importance of the genera Salix L. (willows) and Populus L. (poplars, cottonwoods, and aspens) Salicaceae, we know little about the sources of differences in species diversity between the genera and of the phylogenetic conflict that often confounds estimating phylogenetic trees. Salix subgenera and sections, in particular, have been difficult to classify, with one recent attempt termed a "spectacular failure" due to a speculated radiation of the subgenera Vetrix and Chamaetia. Here, we use targeted sequence capture to understand the evolutionary history of this portion of the Salicaceae plant family. Our phylogenetic hypothesis was based on 787 gene regions and identified extensive phylogenetic conflict among genes. Our analysis supported some previously described subgeneric relationships and confirmed the polyphyly of others. Using an fbranch analysis, we identified several cases of hybridization in deep branches of the phylogeny, which likely contributed to discordance among gene trees. In addition, we identified a rapid increase in diversification rate near the origination of the Vetrix-Chamaetia clade in Salix. This region of the tree coincided with several nodes that lacked strong statistical support, indicating a possible increase in incomplete lineage sorting due to rapid diversification. The extraordinary level of both recent and ancient hybridization in both Salix and Populus have played important roles in the diversification and diversity in these two genera.

The sex determination gene of Populus deltoides, PdFERR, interacts with ERF96 to promote the development of female flower organs.

The female-specifically expressed response regulator (PdFERR) gene in Populus deltoides, a sex determination gene (an orthologous gene of ARR17 in Populus tremula), was found to promote femaleness in heterologous expression lines of Arabidopsis. None of the genes in the Arabidopsis genome seem to be orthologous to PdFERR. Although originating from two evolutionarily distant plants, the dioecious poplar FERR might promote femaleness in the hermaphroditic Arabidopsis through an evolutionary consistent regulatory pathway. However, there is no molecular evidence to support this viewpoint. In this study, to identify the shared downstream orthologous gene of PdFERR, we used yeast two-hybrid assay to screen potential interactors of PdFERR in Arabidopsis. We identified the ethylene response factor 96 (AtERF96) and confirmed the interaction via in vivo and in vitro assays. The ERF96 orthologous gene in P. deltoides was also experimentally confirmed to interact with PdFERR. PdFERR could then promote femaleness in poplar or Arabidopsis through interactions with ERF96, which provide a new perspective for understanding the PdFERR gene regulating sex differentiation.

Design, synthesis and biological evaluation of novel modified dual-target shikonin derivatives for colorectal cancer treatment.

Warburg effect provides energy and material essential for tumor proliferation, the reverse of Warburg effect provides insights into the development of a novel anti-cancer strategy. Pyruvate kinase 2 (PKM2) and pyruvate dehydrogenase kinase 1 (PDK1) are two key enzymes in tumor glucose metabolism pathway that not only contribute to the Warburg effect through accelerating aerobic glycolysis, but also serve as druggable target for colorectal cancer (CRC). Considering that targeting PKM2 or PDK1 alone does not seem to be sufficient to remodel abnormal glucose metabolism and achieve significant antitumor activity, a series of novel benzenesulfonyl shikonin derivatives were designed to regulate PKM2 and PDK1 simultaneously. By means of molecular docking and antiproliferative screen, we found that compound Z10 could act as the combination of PKM2 activator and PDK1 inhibitor, thereby significantly inhibited glycolysis that reshaping tumor metabolism. Moreover, Z10 could inhibit proliferation, migration and induce apoptosis in CRC cell HCT-8. Finally, the in vivo anti-tumor activity of Z10 was evaluated in a colorectal cancer cell xenograft model in nude mice and the results demonstrated that Z10 induced tumor cell apoptosis and inhibited tumor cell proliferation with lower toxicity than shikonin. Our findings indicated that it is feasible to alter tumor energy metabolism through multi-target synergies, and the dual-target benzenesulfonyl shikonin derivative Z10 could be a potential anti-CRC agent.

Genome-Wide Comparative Analysis of the Fasciclin-like Arabinogalactan Proteins (FLAs) in Salicacea and Identification of Secondary Tissue Development-Related Genes.

Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs) containing both AGP-like glycated domains and fasciclin (FAS) domains, which are involved in plant growth and development and synthesis of the cell wall. However, these proteins have not been identified or analyzed in willow, Salix, the sister genus of Populus. In this study, we performed a whole genome study of the FLA gene family of Salix suchowensis and compared it with the FLA gene family of Populus deltoides. The results showed the presence of 40 and 46 FLA genes in P. deltoides and S. suchowensis, distributed on 17 and 16 chromosomes, respectively. Four pairs of tandem repeat genes were found in willow, while poplar had no tandem repeat genes. Twelve and thirteen pairs of duplicated gene fragments were identified in poplar and willow, respectively. The multispecies phylogenetic tree showed that the FLA gene family could be divided into four groups (I-IV), with Group 1 showing significant expansion in woody plants. A gene expression analysis showed that PdeFLA19/27 in Group I of poplar was highly expressed, specifically during the secondary growth period of the stem and the rapid elongation of seed hairs. In the Group I genes of S. suchowensis, SsuFLA25/26/28 was also highly expressed during the secondary growth period, whereas increased expression of SsuFLA35 was associated with seed hair tissue. These results provide important clues about the differences in the FLA gene family during the evolution of herbs and woody plants, and suggest that the FLA gene family may play an essential role in regulating the secondary growth of woody plants. It also provides a reference for further studies on the regulation of secondary growth and seed hair development by FLA genes in poplar and willow.

Genome-Wide Analysis of the Expansin Gene Family in Populus and Characterization of Expression Changes in Response to Phytohormone (Abscisic Acid) and Abiotic (Low-Temperature) Stresses.

Expansins are a group of cell wall enzyme proteins that help to loosen cell walls by breaking hydrogen bonds between cellulose microfibrils and hemicellulose. Expansins are essential plant proteins that are involved in several key processes, including seed germination, the growth of pollen tubes and root hairs, fruit ripening and abscission processes. Currently, there is a lack of knowledge concerning the role of expansins in woody plants. In this study, we analyzed expansin genes using Populus genome as the study target. Thirty-six members of the expansin gene family were identified in Populus that were divided into four subfamilies (EXPA, EXPB, EXLA and EXLB). We analyzed the molecular structure, chromosome localization, evolutionary relationships and tissue specificity of these genes and investigated expression changes in responses to phytohormone and abiotic stresses of the expansin genes of Populus tremula L. (PtEXs). Molecular structure analysis revealed that each PtEX protein had several conserved motifs and all of the PtEXs genes had multiple exons. Chromosome structure analysis showed that the expansin gene family is distributed on 14 chromosomes. The PtEXs gene family expansion patterns showed segmental duplication. Transcriptome data of Populus revealed that 36 PtEXs genes were differently expressed in different tissues. Cis-element analysis showed that the PtEXs were closely associated with plant development and responses to phytohormone and abiotic stress. Quantitative real-time PCR showed that abscisic acid (ABA) and low-temperature treatment affected the expression of some PtEXs genes, suggesting that these genes are involved in responses to phytohormone and abiotic stress. This study provides a further understanding of the expansin gene family in Populus and forms a basis for future functional research studies.

Expression Quantitative Trait Locus of Wood Formation-Related Genes in Salix suchowensis.

Shrub willows are widely planted for landscaping, soil remediation, and biomass production, due to their rapid growth rates. Identification of regulatory genes in wood formation would provide clues for genetic engineering of willows for improved growth traits on marginal lands. Here, we conducted an expression quantitative trait locus (eQTL) analysis, using a full sibling F1 population of Salix suchowensis, to explore the genetic mechanisms underlying wood formation. Based on variants identified from simplified genome sequencing and gene expression data from RNA sequencing, 16,487 eQTL blocks controlling 5505 genes were identified, including 2148 cis-eQTLs and 16,480 trans-eQTLs. eQTL hotspots were identified, based on eQTL frequency in genomic windows, revealing one hotspot controlling genes involved in wood formation regulation. Regulatory networks were further constructed, resulting in the identification of key regulatory genes, including three transcription factors (JAZ1, HAT22, MYB36) and CLV1, BAM1, CYCB2;4, CDKB2;1, associated with the proliferation and differentiation activity of cambium cells. The enrichment of genes in plant hormone pathways indicates their critical roles in the regulation of wood formation. Our analyses provide a significant groundwork for a comprehensive understanding of the regulatory network of wood formation in S. suchowensis.

Genome-Wide Comparison and Functional Characterization of HMGR Gene Family Associated with Shikonin Biosynthesis in Lithospermum erythrorhizon.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), as the rate-limiting enzyme in the mevalonate pathway, is essential for the biosynthesis of shikonin in Lithospermum erythrorhizon. However, in the absence of sufficient data, the principles of a genome-wide in-depth evolutionary exploration of HMGR family members in plants, as well as key members related to shikonin biosynthesis, remain unidentified. In this study, 124 HMGRs were identified and characterized from 36 representative plants, including L. erythrorhizon. Vascular plants were found to have more HMGR family genes than nonvascular plants. The phylogenetic tree revealed that during lineage and species diversification, the HMGRs evolved independently and intronless LerHMGRs emerged from multi-intron HMGR in land plants. Among them, Pinus tabuliformis and L. erythrorhizon had the most HMGR gene duplications, with 11 LerHMGRs most likely expanded through WGD/segmental and tandem duplications. In seedling roots and M9 cultured cells/hairy roots, where shikonin biosynthesis occurs, LerHMGR1 and LerHMGR2 were expressed significantly more than other genes. The enzymatic activities of LerHMGR1 and LerHMGR2 further supported their roles in catalyzing the conversion of HMG-CoA to mevalonate. Our findings provide insight into the molecular evolutionary properties and function of the HMGR family in plants and a basis for the genetic improvement of efficiently produced secondary metabolites in L. erythrorhizon.

A General Model to Explain Repeated Turnovers of Sex Determination in the Salicaceae.

Dioecy, the presence of separate sexes on distinct individuals, has evolved repeatedly in multiple plant lineages. However, the specific mechanisms by which sex systems evolve and their commonalities among plant species remain poorly understood. With both XY and ZW sex systems, the family Salicaceae provides a system to uncover the evolutionary forces driving sex chromosome turnovers. In this study, we performed a genome-wide association study to characterize sex determination in two Populus species, P. euphratica and P. alba. Our results reveal an XY system of sex determination on chromosome 14 of P. euphratica, and a ZW system on chromosome 19 of P. alba. We further assembled the corresponding sex-determination regions, and found that their sex chromosome turnovers may be driven by the repeated translocations of a Helitron-like transposon. During the translocation, this factor may have captured partial or intact sequences that are orthologous to a type-A cytokinin response regulator gene. Based on results from this and other recently published studies, we hypothesize that this gene may act as a master regulator of sex determination for the entire family. We propose a general model to explain how the XY and ZW sex systems in this family can be determined by the same RR gene. Our study provides new insights into the diversification of incipient sex chromosomes in flowering plants by showing how transposition and rearrangement of a single gene can control sex in both XY and ZW systems.

Shikonin N-benzyl matrinic acid ester derivatives as novel telomerase inhibitors with potent activity against lung cancer cell lines.

In this study, a series of novel shikonin N-benzyl matrinic acid ester derivatives (PMMB-299-PMMB-310) were synthesized and tested for their ability to inhibit the proliferation of cancer cells. Compared with shikonin and matrine, some of the ester derivatives were found to exhibit better anti-proliferative activity against seven different cancer cell lines, with less cytotoxicity toward non-cancerous cells. The strongest anti-proliferative activity was exhibited by PMMB-302, which had an IC50 value of 2.71 µM against A549 cells. The compound caused cell cycle arrest in the G2/M phase and induced apoptosis. Effects on the expression of apoptosis-related molecules such as Bcl2, Bcl-XL, caspase-3, caspase-9 and FADD suggested that PMMB-302 has tumor suppressive roles in lung cancer cells. In addition, PMMB-302 inhibited expression of telomerase core proteins, dyskerin and NHP2, and telomerase reverse transcriptase RNA. Moreover, molecular docking of PMMB-302 was subsequently conducted to determine the probable binding mode with telomerase. Taken together, the results indicate that PMMB-302 acts as a tumor suppressor in lung cancer cells by negatively regulating telomerase expression.

First Report of Diaporthe cercidis and D. nobilis Causing Leaf Blotch on Populus davidiana × P. bolleana in China.

Shanxin yang (Populus davidiana × P. bolleana) is a commercially valuable hybrid poplar that is widely planted in northern China. Efficient genetic transformation and gene-editing systems have been established for this hybrid poplar (Wang et al., 2011; Wang et al., 2020). However, records of fungal diseases on Shanxin yang are very limited. In July 2020, large necrotic lesions were observed on 16 one-year-old Shanxin yang seedlings planted in a greenhouse of Nanjing Forestry University, Nanjing, China. The disease symptoms appeared mostly on the leaves and not on the stems. Symptoms first manifested as differently sized and shaped brown spots, having clearly demarcated margins. As the disease progressed, the spots coalesced, and large lesions were present on the leaves. Severe infections resulted in whole-plant defoliation and eventually plant death. Small leaf samples (5 mm2) cut from lesion margins were surface sterilized with 75% ethanol for 30 s, followed by 1% NaClO for 90 s and then washed three times with sterile distilled water. After drying on sterilized filter paper, the cut tissues were plated on potato dextrose agar (PDA) supplemented with ampicillin (100 µg/mL) and incubated at 25°C in the dark. Three isolates (named as SX-1, SX-2 and SX-3, respectively) were obtained after 5 days. The isolation frequency was low, which might be due to the greenhouse microclimate that was unfavorable for pathogen spread. Mycelial plugs (5 mm) cut from the leading edge of the mycelia were transferred onto fresh PDA and synthetic nutrient-poor agar (SNA) plates to obtain pure cultures. On both media, colonies of the isolates were white on the front and light yellowish on the back, with concentric zonation. Abundant aerial mycelia developed; the hyphae were hyaline, non-septate, and approximately 0.794-2.961 µm in diameter. On the SNA medium, SX-1 and SX-3 produced globose to subglobose, black pycnidia after 18 days under a 12 h photoperiod. The alpha conidia were fusoid, aseptate, hyaline, smooth, and 6.4 ± 1.2 × 2.4 ± 0.6 µm (n = 50) in size. Under the same conditions, SX-2 produced pycnidia after 20 days, and the conidia were 2.8 ± 0.7 × 7.5 ± 1.3 µm. Beta conidia were not observed on any colony. Based on the morphological characteristics, the isolated mycelia resembled Diaporthe spp. (Gomes et al., 2013). To determine the species identity, genomic DNA from each isolate was extracted, and five loci were amplified, namely, part of the internal transcribed spacer (ITS) amplified with primers ITS1/ITS4 (White et al. 1990); part of the translation elongation factor 1-alpha (EF1-α) with primers EF1-728F/EF1-986R (Carbone and Kohn, 1999); part of the calmodulin (CAL) with primers CAL-228F/CAL-737R (Carbone and Kohn, 1999); part of the ß-tubulin (ß-tub) with primers Bt2a/Bt2b (Glass and Donaldson, 1995), and part of the histone H3 (HIS) with primers CYLH3F/H3-1b (Glass and Donaldson 1995, Crous et al., 2004). The obtained sequences were deposited in GenBank (accession numbers are listed in Table S1). BLAST analyses showed that the all the amplified fragments were highly homologous to Diaporthe spp. (Table S1). Based on concatenated sequences of the amplicons, a phylogenetic tree was constructed by using Maximum-likelihood and Bayesian inference methods. The multi-locus phylogenetic analyses distinguished SX-1 and SX-3 as D. cercidis, and SX-2 as D. nobilis. To complete Koch's postulates, the pathogenicity of SX-1, as well as SX-2, was tested on both detached and attached leaves of one-year-old Shanxin yang seedlings grown under greenhouse conditions. Healthy leaves were pierced with a sterile needle and then inoculated independently with 5-mm mycelial plugs cut from the edge of the 4-day-old colonies of SX-1 and SX-2 colonies. Controls were inoculated with noncolonized PDA plugs. Three replicates were prepared for each isolate. For the in-vitro tests, detached leaves were placed on wet filter paper in parafilm-sealed Petri dishes and cultured at 25 °C in the dark. For the attached leaf assays, the plants were kept in an 85% humidity chamber immediately after inoculation. All the inoculated leaves developed dark or brown necrotic lesions at 5 days after inoculation, whereas the control leaves showed no symptoms. D. cercidis and D. nobilis were separately reisolated from the inoculated leaves. The former was first described by Yang et al. (2018) as occurring on twigs and branches of Cercis chinensis, and very recently, this pathogen was reported to cause leaf blotch on Acer pictum subsp. mono (Wan et al., 2021). The latter infects some fruit trees (Yu et al., 2018; Sun et al., 2019; Ma et al., 2019) and chestnut (Zhang et al., 2018). All of these studies were conducted in China where there is a great diversity of Diaporthe species (Yang et al., 2018). To our knowledge, this is the first report of both D. cercidis and D. nobilis causing leaf blotch on poplar. The identification of these pathogens is essential for understanding the range of their host species and to manage the resulting fungal diseases, which may cause severe economic damage.

The genomic architecture of the sex-determining region and sex-related metabolic variation in Ginkgobiloba.

Sex differences and evolutionary differences are critical biological issues. Ginkgo is an ancient lineage of dioecious gymnosperms with special value for studying the mechanism of sex determination in plants. However, the major genetic basic underlying sex chromosomes remains to be uncovered. In this study, we identify the sex-determining region of Ginkgo and locate it to the area from megabases 48 to 75 on chromosome 2. We find that the male sex-determining region of Ginkgo contains more than 200 genes, including four MADS-box genes, demonstrating that the Ginkgo sex determination system is of the XY type. We also find that genetic sex differences result in specialized flavonoid metabolism and regulation in each sex. These findings establish a foundation for revealing the molecular mechanism of sexual dimorphism and promoting the development of the Ginkgo industry.

The Acer truncatum genome provides insights into nervonic acid biosynthesis.

Acer truncatum (purpleblow maple) is a woody tree species that produces seeds with high levels of valuable fatty acids (especially nervonic acid). However, the lack of a complete genome sequence has limited both basic and applied research on A. truncatum. We describe a high-quality draft genome assembly comprising 633.28 Mb (contig N50 = 773.17 kb; scaffold N50 = 46.36 Mb) with at least 28 438 predicted genes. The genome underwent an ancient triplication, similar to the core eudicots, but there have been no recent whole-genome duplication events. Acer yangbiense and A. truncatum are estimated to have diverged about 9.4 million years ago. A combined genomic, transcriptomic, metabonomic, and cell ultrastructural analysis provided new insights into the biosynthesis of very long-chain monounsaturated fatty acids. In addition, three KCS genes were found that may contribute to regulating nervonic acid biosynthesis. The KCS paralogous gene family expanded to 28 members, with 10 genes clustered together and distributed in the 0.27-Mb region of pseudochromosome 4. Our chromosome-scale genomic characterization may facilitate the discovery of agronomically important genes and stimulate functional genetic research on A. truncatum. Furthermore, the data presented also offer important foundations from which to study the molecular mechanisms influencing the production of nervonic acids.

Sex determination through X-Y heterogamety in Salix nigra.

The development of non-recombining sex chromosomes has radical effects on the evolution of discrete sexes and sexual dimorphism. Although dioecy is rare in plants, sex chromosomes have evolved repeatedly throughout the diversification of angiosperms, and many of these sex chromosomes are relatively young compared to those found in vertebrates. In this study, we designed and used a sequence capture array to identify a novel sex-linked region (SLR) in Salix nigra, a basal species in the willow clade, and demonstrated that this species has XY heterogamety. We did not detect any genetic overlap with the previously characterized ZW SLRs in willows, which map to a different chromosome. The S. nigra SLR is characterized by strong recombination suppression across a 2 MB region and an excess of low-frequency alleles, resulting in a low Tajima's D compared to the remainder of the genome. We speculate that either a recent bottleneck in population size or factors related to positive or background selection generated this differential pattern of Tajima's D on the X and autosomes. This discovery provides insights into factors that may influence the evolution of sex chromosomes in plants and contributes to a large number of recent observations that underscore their dynamic nature.

Design, synthesis and biological evaluation of anilide (dicarboxylic acid) shikonin esters as antitumor agents through targeting PI3K/Akt/mTOR signaling pathway.

Triple-negative breast cancer (TNBC) has an unfavorable prognosis attribute to its low differentiation, rapid proliferation and high distant metastasis rate. PI3K/Akt/mTOR as an intracellular signaling pathway plays a key role in the cell proliferation, migration, invasion, metabolism and regeneration. In this work, we designed and synthesized a series of anilide (dicarboxylic acid) shikonin esters targeting PI3K/Akt/mTOR signaling pathway, and assessed their antitumor effects. Through three rounds of screening by computer-aided drug design method (CADD), we preliminarily obtained sixteen novel anilide (dicarboxylic acid) shikonin esters and identified them as excellent compounds. CCK-8 assay results demonstrated that compound M9 exhibited better antiproliferative activities against MDA-MB-231, A549 and HeLa cell lines than shikonin (SK), especially for MDA-MB-231 (M9: IC50 = 4.52 ± 0.28 µM; SK: IC50 = 7.62 ± 0.26 µM). Moreover, the antiproliferative activity of M9 was better than that of paclitaxel. Further pharmacological studies showed that M9 could induce apoptosis of MDA-MB-231 cells and arrest the cell cycle in G2/M phase. M9 also inhibited the migration of MDA-MB-231 cells by inhibiting Wnt/ß-catenin signaling pathway. In addition, western blot results showed that M9 could inhibit cell proliferation and migration by down-regulating PI3K/Akt/mTOR signaling pathway. Finally, a three-dimensional quantitative structure-activity relationship (3D-QSAR) model was also constructed to provide a basis for further development of shikonin derivatives as potential antitumor drugs through structure-activity relationship analysis. To sum up, M9 could be a potential candidate for TNBC therapy.

Phylogenomics of the genus Populus reveals extensive interspecific gene flow and balancing selection.

Phylogenetic analysis is complicated by interspecific gene flow and the presence of shared ancestral polymorphisms, particularly those maintained by balancing selection. In this study, we aimed to examine the prevalence of these factors during the diversification of Populus, a model tree genus in the Northern Hemisphere. We constructed phylogenetic trees of 29 Populus taxa using 80 individuals based on re-sequenced genomes. Our species tree analyses recovered four main clades in the genus based on consensus nuclear phylogenies, but in conflict with the plastome phylogeny. A few interspecific relationships remained unresolved within the multiple-species clade because of inconsistent gene trees. Our results indicated that gene flow has been widespread within each clade and also occurred among the four clades during their early divergence. We identified 45 candidate genes with ancient polymorphisms maintained by balancing selection. These genes were mainly associated with mating compatibility, growth and stress resistance. Both gene flow and selection-mediated ancient polymorphisms are prevalent in the genus Populus. These are potentially important contributors to adaptive variation. Our results provide a framework for the diversification of model tree genus that will facilitate future comparative studies.

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.

IGDD: a database of intronless genes in dicots.

BACKGROUND: Intronless genes are a significant characteristic of prokaryotes. Systematic identification and annotation are primary and crucial steps for determining the functions of intronless genes and understanding their occurrence in eukaryotes. DESCRIPTION: In this paper, we describe the construction of the Intronless Genes Database in Dicots (IGDD; available at http://bio.njfu.edu.cn/igdd/ ), which contains data for five well-annotated plants including Arabidopsis thaliana, Carica papaya, Populus trichocarpa, Salix suchowensis and Vitis vinifera. Using highly visual settings, IGDD displays the structural and functional annotations, the homolog groups, the syntenic relationships, the expression patterns, and the statistical characteristics of intronless genes. In addition, useful tools such as an advanced search and local BLAST are available through a user-friendly and intuitive web interface. CONCLUSION: In conclusion, the IGDD provides a comprehensive and up-to-date platform for researchers to assist the exploration of intronless genes in dicot plants.

High-level SUMO-mediated fusion expression of ABP-dHC-cecropin A from multiple joined genes in Escherichia coli.

The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, evaluation of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli. To improve the expression level of ABP-dHC-cecropin A in E. coli, tandem repeats of the ABP-dHC-cecropin A gene were constructed and expressed as fusion proteins (SUMO-nABP-dHC-cecropin, n = 1, 2, 3, 4) via pSUMO-nABP-dHC-cecropin A vectors (n = 1, 2, 3, 4). Comparison of the expression levels of soluble SUMO-nABP-dHC-cecropin A fusion proteins (n = 1, 2, 3, 4) suggested that BL21 (DE3)/pSUMO-3ABP-dHC-cecropin A is an ideal recombinant strain for ABP-dHC-cecropin A production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of ABP-dHC-cecropin A was as high as 65 mg/L, with ∼21.3% of the fusion protein in soluble form. By large-scale fermentation, protein production reached nearly 300 mg/L, which is the highest yield of ABP-dHC-cecropin A reported to date. In antibacterial experiments, the efficacy was approximately the same as that of synthetic ABP-dHC-cecropin A. This method provides a novel and effective means of producing large amounts of ABP-dHC-cecropin A.

RNA-directed DNA methylation in plants.

In plants, many small interfering RNAs (siRNAs) direct de novo methylation by DNA methyltransferase. DNA methylation typically occurs by RNA-directed DNA methylation (RdDM), which directs transcriptional gene silencing of transposons and endogenous transgenes. RdDM is driven by non-coding RNAs (ncRNAs) produced by DNA-dependent RNA polymerases IV and V (PolIV and PolV). The production of siRNAs is initiated by PolIV and ncRNAs produced by PolIV are precursors of 24-nucleotide siRNAs. In contrast, ncRNAs produced by PolV are involved in scaffolding RNAs. In this review, we summarize recent studies of RdDM. In particular, we focus on the mechanisms involved in chromatin remodeling by PolIV and PolV.

[Advances in lineage-specific genes].

Lineage-specific genes (LSGs) are defined as genes found in one particular taxonomic group but have no significant sequence similarity with genes from other lineages, which compose about 10%?20% of the total genes in the genome of a focal organism. LSGs were first uncovered in the yeast genome in 1996. The development of the whole genome sequencing leads to the emergence of studies on LSGs as a hot topic in comparative genomics. LSGs have been extensively studied on microbial species, lower marine organisms, plant (such as Arabidopsis thaliana, Oryza sativa, Populus), insects, primate, etc; the biological functions of LSGs are important to clarify the evolution and adaptability of a species. In this review, we summarize the progress of LSGs studies, including LSGs identification, gene characterization, origin and evolution of LSGs, biological function, and expression analysis of LSGs. In addition, we discuss the existing problems and future directions for studies in this area. Our purpose is to provide some unique insights into the researches of LSGs.