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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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Gene expression analysis is crucial for uncovering components underlying important biological processes for a focal organism. Large-scale gene co-expression networks generally exhibit small-world, scale-free properties; and the degree distributions of these networks follow power-law forms. Topology properties are often informative for determining the key components of the biological systems and their genetic mechanisms. Some basic topology properties display dissimilarity in different tissues, which helps to elucidate the different genetic mechanisms underlying important biological processes among tissues from the top to bottom of trees. In this study, the topology properties of gene co-expression networks were compared in leaf, shoot, wood, and root tissues of poplar. The comparison results demonstrated that the differences of topology properties exist among tissues and the root tissue displays larger average degree and network density, indicating that genes in root tissue are more highly co-expressed than those in the other three tissues. The nodes with a large degree, also known as hub genes, were annotated by the NetAffx Analysis Center and the agriGO tool, with annotation results that these highly interconnected genes are involved in the key biological processes in each tissue. This study also revealed the topology properties' differences between gene co-expression networks and random network, suggesting the existence of hierarchically organized module and small-world organizations in networks of poplar. The approach described in this research offers an effective strategy for identifying key genes involved in the important biological processes in poplar. [ABSTRACT FROM AUTHOR]

Gene knockdown and gene-silencing pathways in eukaryotic organisms are associated with small RNAs 20 to 25 nucleotides in length, which include microRNAs (miRNAs) and small interfering RNAs (siRNAs). These small RNAs are recruited to repress gene expression upstream or downstream of the transcription pathway. RNA interference (RNAi) is a biological inhibitor of gene expression that results in the destruction of messenger RNAs (mRNAs), leading to the inhibition of protein production. Indeed, RNA silencing plays a key role in plant development in terms of the plant’s response to both biotic and abiotic stresses. Conversely, Viral Suppressors of RNA silencing (VSRs) are proteins that hamper antiviral RNAi activation in plants, lead to suppress plant RNA-silencing. These VSR proteins prevent the induction of the plant antiviral RNAi immune response. This review focuses on small RNAs in plants and their roles in the responses of plants to biotic and abiotic stresses. [ABSTRACT FROM AUTHOR]

The huge amount of gene expression data generated by microarray and next-generation sequencing technologies present challenges to exploit their biological meanings. When searching for the coexpression genes, the data mining process is largely affected by selection of algorithms. Thus, it is highly desirable to provide multiple options of algorithms in the user-friendly analytical toolkit to explore the gene expression signatures. For this purpose, we developed GESearch, an interactive graphical user interface (GUI) toolkit, which is written in MATLAB and supports a variety of gene expression data files. This analytical toolkit provides four models, including the mean, the regression, the delegate, and the ensemble models, to identify the coexpression genes, and enables the users to filter data and to select gene expression patterns by browsing the display window or by importing knowledge-based genes. Subsequently, the utility of this analytical toolkit is demonstrated by analyzing two sets of real-life microarray datasets from cell-cycle experiments. Overall, we have developed an interactive GUI toolkit that allows for choosing multiple algorithms for analyzing the gene expression signatures. [ABSTRACT FROM AUTHOR]

Salix is a dioecious plant. Research on the molecular regulation mechanism of male and female inflorescence differentiation and development is necessary to analyze sex differentiation in the willow and the underlying mechanisms of unisexual flower development. However, at present, there are no reference genes suitable for stable expression in the process of willow inflorescence development. In this study, Salix suchowensis was used as the research material, nine candidate reference genes (α-TUB1, α-TUB2, ACT, H2A, DnaJ, CDC2, GAPDH, TIP41, β-TUB) were selected, and qRT-PCR technology was used to detect the expression of each candidate reference gene in female and male flowers at different developmental stages and using five algorithms (geNorm, Normfinder, Delta Ct, BestKeeper, and RefFinder) to comprehensively evaluate the stability of candidate reference genes. The results showed that ACT and DnaJ were stably expressed in all samples and could be used as reference genes. In addition, the reliability of the screening results was further verified via an expression pattern analysis of the CFS gene that encodes flower specific transcription factor in different samples. The stable reference genes selected in this study provide the basis for future research on the expression analysis of functional genes related to the development of male and female flowers of S. suchowensis. [ABSTRACT FROM AUTHOR]

Background: Variegation in flower color is commonly observed in many plant species and also occurs on ornamental peaches (Prunus persica f. versicolor [Sieb.] Voss). Variegated plants are highly valuable in the floricultural market. To gain a global perspective on genes differentially expressed in variegated peach flowers, we performed large-scale transcriptome sequencing of white and red petals separately collected from a variegated peach tree. Results: A total of 1,556,597 high-quality reads were obtained, with an average read length of 445 bp. The ESTs were assembled into 16,530 contigs and 42,050 singletons. The resulting unigenes covered about 60% of total predicted genes in the peach genome. These unigenes were further subjected to functional annotation and biochemical pathway analysis. Digital expression analysis identified a total of 514 genes differentially expressed between red and white flower petals. Since peach flower coloration is determined by the expression and regulation of structural genes relevant to flavonoid biosynthesis, a detailed examination detected four key structural genes, including C4H, CHS, CHI and F3H, expressed at a significantly higher level in red than in white petal. Except for the structural genes, we also detected 11 differentially expressed regulatory genes relating to flavonoid biosynthesis. Using the differentially expressed structural genes as the test objects, we validated the digital expression results by using quantitative real-time PCR, and the differential expression of C4H, CHS and F3H were confirmed. Conclusion: In this study, we generated a large EST collection from flower petals of a variegated peach. By digital expression analysis, we identified an informative list of candidate genes associated with variegation in peach flowers, which offered a unique opportunity to uncover the genetic mechanisms underlying flower color variegation. [ABSTRACT FROM AUTHOR]

qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate. [ABSTRACT FROM AUTHOR]

Pinus massoniana and P. hwangshanensis are two conifer species located in southern China, which are of both economic and ornamental value. Around the middle and lower reaches of the Yangtze River, P. massoniana occurs mainly at altitudes below 700 m, while P. hwangshanensis can be found above 900 m. At altitudes where the distribution of both pines overlaps, a natural introgression hybrid exists, which we will further refer to as the Z pine. This pine has a morphological character that shares attributes of both P. massoniana and P. hwangshanensis. However, compared to the other two pines, its reproductive structure, the pinecone, has an ultra-low ripening rate with seeds that germinate poorly. In this study, we aimed to find the reason for the impaired cone maturation by comparing transcriptome libraries of P. massoniana and Z pine cones at seven successive growth stages. After sequencing and assembly, we obtained unigenes and then annotated them against NCBI's non-redundant nucleotide and protein sequences, Swiss-Prot, Clusters of Orthologous Groups, Gene Ontology and KEGG Orthology databases. Gene expression levels were estimated and differentially expressed genes (DEGs) of the two pines were mined and analyzed. We found that several of them indeed relate to reproductive process. At every growth stage, these genes are expressed at a higher level in P. massoniana than in the Z pine. These data provide insight into understanding which molecular mechanisms are altered between P. massoniana and the Z pine that might cause changes in the reproductive process. [ABSTRACT FROM AUTHOR]

Cytokinins have vital effects on various plant development-related processes. Type-A response regulators (RRs) are among the most important regulators in the cytokinin signaling pathway. Specifically, RR genes are the primary cytokinin-responsive genes, encode negative regulators of cytokinin signaling. Similar sex-determining mechanisms involving a type-A RR homolog have recently been identified in sister genera Populus and Salix. Here, we aimed to identify and characterize type-A RR gene family members in Populus deltoides. On the basis of the conserved receiver domain and phylogenetic analysis, we identified 9, 11, and 14 type-A RR genes were identified in P. deltoides , Populus trichocarpa , and Salix suchowensis , respectively. The exon-intron organization and chromosomal locations of these genes in P. deltoides as well as the characteristics of the encoded proteins were analyzed in detail. Gene duplication events, molecular evolution, and phylogenetic relationships were analyzed for all identified type-A RR genes along with 10 Arabidopsis thaliana homologs. These results indicated that the expansion of the type-A RR gene family was primarily due to whole-genome and segmental duplication events. The collinear type-A ARR pairs in A. thaliana were all derived from the α-whole-genome duplication (WGD) event, whereas those in the three Salicaceae species were divided into two groups, one comprising 10 pairs derived from the p -WGD event and the other consisting of 11 pairs derived from the γ-WGD event or a more ancient duplication event. All of the duplicated paralogous gene pairs had undergone purifying selection. Although different type-A RR paralogs experienced different selection pressures, convergent evolution was detected within the receiver domain-encoding sequence, indicative of the importance of this domain for the phosphorelay-related function of the encoded proteins. Three type-A RR genes (PdRR9 , PtRR11 , and SsRR12) associated with sex determination in Salicaceae species formed a small subclade that lacked A. thaliana counterparts. Evaluation of gene expression in vegetative tissues and at different floral developmental stages in P. deltoides revealed the specific spatiotemporal expression of each PdRR gene, with two (PdRR8 and PdRR9) of the nine PdRR genes exhibiting the opposite sex-biased expression patterns. Diverse expression levels were detected between all eight paralogous PdRR pairs. These findings support future research to elucidate the functional divergence of type-A RR genes. • Systematic genome-wide analysis of type-A RR genes was conducted in P. deltoides. • Expansion of the type-A RR gene family was primarily due to the WGD/segmental duplication events. • The PdRRs exhibited distinct developmental stage- and tissue-specific expression patterns. • This study will provide critical information to further elucidate the functional divergence of type-A RR genes. [ABSTRACT FROM AUTHOR]