Comparative transcriptome combined with biochemical and physiological analyses provide new insights toward cadmium accumulation with two contrasting Nicotiana species.

Cadmium (Cd) is known as one of the most hazardous elements in the environment and a persistent soil constraint toxic to all flora and fauna. In this study, we conducted physiological, biochemical, and transcriptomic analyses of Nicotiana rustica (N. rustica) and Nicotiana tabacum (N. tabacum) treated with CdCl2 to know the underlying molecular mechanisms of Cd accumulation. As a result, N. rustica had more dry weight than N. tabacum. Additionally, N. rustica accumulated higher Cd concentration (69.65 times), Cd2+ influx (1.32-fold), glutathione S-transferases (GST) enzyme activity (2.54 times), GSH/GSSG (oxidized form of GSH) ratio, increase of superoxide dismutase and CAT and a lower H2 O2 and superoxide (O2 •- ) accumulation in their roots than N. tabacum. Cd mainly distributed in the cytoplasm of both species and N. rustica had a significant proportion in the cell wall. Furthermore, the transcriptomic analysis revealed 173 and 710 differentially expressed genes (DEGs) between control and Cd-stressed plants in the leaves and roots of N. rustica, while 576 and 1543 DEGs were found in the leaves and roots of N. tabacum, respectively. In N. rustica, phenylpropanoid biosynthesis and phenylalanine metabolism were the most enriched pathways, while GSH metabolism, ATP-binding cassette transporters and phenylpropanoid biosynthesis were the most enriched in N. tabacum. Finally, we found that DEGs related to metal influx, sequestration, remobilization, and chelation were responsible for Cd accumulation. These results indicated that N. rustica accumulated higher Cd content than N. tabacum, suggesting that each species utilized different response mechanism under the same Cd stress conditions. The DEGs identified in this study might lead to the identification of genes or pathways related to Cd regulation. This study identifies important regulators related to Cd accumulation.

The evolution of opsin genes in five species of mirid bugs: duplication of long-wavelength opsins and loss of blue-sensitive opsins.

BACKGROUND: Color vision and phototactic behavior based on opsins are important for the fitness of insects because of their roles in foraging and mate choice. Related topics, including the duplication and loss of opsin genes, have been well investigated in insect orders such as Coleoptera, Lepidoptera, Hymenoptera, Odonata and Orthoptera, and the findings have been used to develop pest management strategies involving light trapping. Mirid bugs of Hemiptera, which are pests that cause heavy economic losses, show capacity for color discrimination and phototaxis. However, the opsins in mirid bugs remain uncharacterized. Herein, we examined five species to investigate the evolution of opsins in the family Miridae. RESULTS: Using RNA-seq, we identified several contigs showing high identity with opsins, including four contigs in Apolygus lucorum and three contigs each in Adelphocoris suturalis, Adelphocoris fasciaticollis, Adelphocoris lineolatus and Nesidiocoris tenuis. Phylogenetic analyses indicated that one of these genes clustered with ultraviolet-sensitive (UV) opsins and that the others clustered with long-wavelength (LW) opsins, suggesting that duplication of LW opsins and loss of blue light-sensitive (B) opsins occurred in mirid bugs. The existence of introns in the LW opsins of mirid bugs suggested that the duplication events were DNA based. Both LW1 and LW2 opsins of mirid bugs were found to be under strong purifying selection. The LW1 opsins were significantly more highly expressed than the LW2 and UV opsins. CONCLUSIONS: We identified the opsins of mirid bugs using five selected mirid species as a representative sample. Phylogenetic analyses clustered one of the genes with UV opsins and the others with LW opsins, suggesting the occurrence of LW opsin duplication and B opsin loss during the evolution of mirid bugs. Intron detection suggested that the identified duplication event was DNA based. The evidence of strong purifying selection and the relatively high expression levels suggested that these opsins exhibit fundamental functions in mirid bugs.

MG2C: a user-friendly online tool for drawing genetic maps.

Genetic map is a linear arrangement of the relative positions of sites in the chromosome or genome based on the recombination frequency between genetic markers. It is the important basis for genetic analysis. Several kinds of software have been designed for genetic mapping, but all these tools require users to write or edit code, making it time-costing and difficult for researchers without programming skills to handle with. Here, MG2C, a new online tool was designed, based on PERL and SVG languages.Users can get a standard genetic map, only by providing the location of genes (or quantitative trait loci) and the length of the chromosome, without writing additional code. The operation interface of MG2C contains three sections: data input, data output and parameters. There are 33 attribute parameters in MG2C, which are further divided into 8 modules. Values of the parameters can be changed according to the users' requirements. The information submitted by users will be transformed into the genetic map in SVG file, which can be further modified by other image processing tools.MG2C is a user-friendly and time-saving online tool for drawing genetic maps, especially for those without programming skills. The tool has been running smoothly since 2015, and updated to version 2.1. It significantly lowers the technical barriers for the users, and provides great convenience for the researchers.

Characterization and Mapping of a Novel Premature Leaf Senescence Mutant in Common Tobacco (Nicotiana tabacum L.).

As the last stage of plant development, leaf senescence has a great impact on plant's life cycle. Genetic manipulation of leaf senescence has been used as an efficient approach in improving the yield and quality of crop plants. Here we describe an ethyl methane sulfonate (EMS) mutagenesis induced premature leaf senescence mutant yellow leaf 1 (yl1) in common tobacco (Nicotiana tabacum L.). The yl1 plants displayed early leaf yellowing. Physiological parameters and marker genes expression indicated that the yl1 phenotype was caused by premature leaf senescence. Genetic analyses indicated that the yl1 phenotype was controlled by a single recessive gene that was subsequently mapped to a specific interval of tobacco linkage group 11 using simple sequence repeat (SSR) markers. Exogenous plant hormone treatments of leaves showed that the yl1 mutant was more sensitive to ethylene and jasmonic acid than the wild type. No similar tobacco premature leaf senescence mutants have been reported. This study laid a foundation for finding the gene controlling the mutation phenotype and revealing the molecular regulation mechanism of tobacco leaf senescence in the next stage.

Insight into the B3Transcription Factor Superfamily and Expression Profiling of B3 Genes in Axillary Buds after Topping in Tobacco(Nicotiana tabacum L.).

Members of the plant-specific B3 transcription factor superfamily play important roles in various growth and developmental processes in plants. Even though there are many valuable studies on B3 genes in other species, little is known about the B3 superfamily in tobacco. We identified 114 B3 proteins from tobacco using comparative genome analysis. These proteins were classified into four subfamilies based on their phylogenetic relationships, and include the ARF, RAV, LAV, and REM subfamilies. The chromosomal locations, gene structures, conserved protein motifs, and sub-cellular localizations of the tobacco B3 proteins were analyzed. The patterns of exon-intron numbers and arrangement and the protein structures of the tobacco B3 proteins were in general agreement with their phylogenetic relationships. The expression patterns of 114 B3 genes revealed that many B3 genes show tissue-specific expression. The expression levels of B3 genes in axillary buds after topping showed that the REM genes are mainly up-regulated in response to topping, while the ARF genes are down-regulated after topping.

Duplication and expression of horizontally transferred polygalacturonase genes is associated with host range expansion of mirid bugs.

BACKGROUD: Horizontal gene transfer and gene duplication are two major mechanisms contributing to the evolutionary adaptation of organisms. Previously, polygalacturonase genes (PGs) were independently horizontally transferred and underwent multiple duplications in insects (e.g., mirid bugs and beetles). Here, we chose three phytozoophagous mirid bugs (Adelphocoris suturalis, A. fasciaticollis, A. lineolatus) and one zoophytophagous mirid bug (Nesidiocoris tenuis) to detect whether the duplication, molecular evolution, and expression levels of PGs were related to host range expansion in mirid bugs. RESULTS: By RNA-seq, we reported 30, 20, 19 and 8 PGs in A. suturalis, A. fasciaticollis, A. lineolatus and N. tenuis, respectively. Interestingly, the number of PGs was significantly positive correlation to the number of host plants (P = 0.0339) in mirid bugs. Most PGs (> 17) were highly expressed in the three phytozoophagous mirid bugs, while only one PG was relatively highly expressed in the zoophytophagous mirid bug. Natural selection analysis clearly showed that a significant relaxation of selection pressure acted on the PGs in zoophytophagous mirid bugs (K = 0.546, P = 0.0158) rather than in phytozoophagous mirid bugs (K = 1, P = 0.92), suggesting a function constraint of PGs in phytozoophagous mirid bugs. CONCLUSION: Taken together with gene duplication, molecular evolution, and expression levels, our results suggest that PGs are more strictly required by phytozoophagous than by zoophytophagous mirid bugs and that the duplication of PGs is associated with the expansion of host plant ranges in mirid bugs.

NAC Family Transcription Factors in Tobacco and Their Potential Role in Regulating Leaf Senescence.

The NAC family is one of the largest families of plant-specific transcription factors (TFs) and NAC proteins play important regulatory roles in a variety of developmental and stress response processes in plants. Members of the NAC family TFs have been shown to be important regulators of leaf senescence in a number of plant species. Here we report the identification of the NAC family in tobacco (Nicotiana tabacum) and characterization of the potential role of some of the tobacco NAC TFs in regulating leaf senescence. A total of 154 NAC genes (NtNACs) were identified and clustered together with the Arabidopsis NAC family into fifteen groups (a-o). Transcriptome data analysis followed by qRT-PCR validation showed that the majority of the senescence-up-regulated NtNACs fall into subgroups NAC-b and f. A number of known senescence regulators from Arabidopsis also belong to these two subgroups. Among these senescence-up-regulated NtNACs, NtNAC080, a close homolog of AtNAP, is a positive regulator of leaf senescence. Overexpression of NtNAC080 caused early senescence in Arabidopsis leaves and NtNAC080 mutation induced by Cas9/gRNA in tobacco led to delayed leaf senescence.

Characterization of a novel RNA virus from Nesidiocoris tenuis related to members of the genus Iflavirus.

The complete genome of a novel virus from Nesidiocoris tenuis was determined by RNA-seq and rapid amplification of cDNA ends. This virus has a single-stranded RNA genome of 10633 nucleotides (nt) in length, not including the poly(A) tail, and contains two putative open reading frames (ORFs). ORF1 encodes a polypeptide of 1320 amino acids (aa) with a predicted molecular mass of 147.92 kDa and theoretical isoelectric point (pI) of 6.96. ORF2 encodes a polypeptide of 1728 aa with a predicted molecular mass of 197.09 kDa and pI of 6.73. Phylogenetic analysis with the deduced aa sequences of the conserved RNA dependent RNA polymerase domain as well as whole genome nt sequences indicated that the virus clusters with viruses classified within the genus Iflavirus, with a high bootstrap value in the maximum-likelihood and neighbor-joining trees. However, this virus has a distinct genome structure with two ORFs, iflaviruses normally having one, suggesting the virus might be a prototype of a new genus. We named the virus isolate Nesidiocoris tenuis virus 1 (NtV-1). The prevalence of NtV-1 infection in wild samples of N. tenuis was at a low level (7.32%, 6 positive in 82 samples), suggesting a possible harmful effect to its host.

Morphological phenotyping and genetic analyses of a new chemical-mutagenized population of tobacco (Nicotiana tabacum L.).

MAIN CONCLUSION: A novel tobacco mutant library was constructed, screened, and characterized as a crucial genetic resource for functional genomics and applied research. A comprehensive mutant library is a fundamental resource for investigating gene functions, especially after the completion of genome sequencing. A new tobacco mutant population induced by ethyl methane sulfonate mutagenesis was developed for functional genomics applications. We isolated 1607 mutant lines and 8610 mutant plants with altered morphological phenotypes from 5513 independent M2 families that consisted of 69,531 M2 plants. The 2196 mutations of abnormal phenotypes in the M2 putative mutants were classified into four groups with 17 major categories and 51 subcategories. More than 60% of the abnormal phenotypes observed fell within the five major categories including plant height, leaf shape, leaf surface, leaf color, and flowering time. The 465 M2 mutants exhibited multiple phenotypes, and 1054 of the 2196 mutations were pleiotropic. Verification of the phenotypes in advanced generations indicated that 70.63% of the M3 lines, 84.87% of the M4 lines, and 95.75% of the M5 lines could transmit original mutant phenotypes of the corresponding M2, M3, and M4 mutant plants. Along with the increased generation of mutants, the ratios of lines inheriting OMPs increased and lines with emerging novel mutant phenotypes decreased. Genetic analyses of 18 stably heritable mutants showed that two mutants were double recessive, five were monogenic recessive, eight presented monogenic dominant inheritance, and three presented semi-dominant inheritance. The pleiotropy pattern, saturability evaluation, research prospects of genome, and phenome of the mutant populations were also discussed. Simultaneously, this novel mutant library provided a fundamental resource for investigating gene functions in tobacco.

Mechanisms and Physiological Roles of the CBL-CIPK Networking System in Arabidopsis thaliana.

Calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CIPK) network is one of the vital regulatory mechanisms which decode calcium signals triggered by environmental stresses. Although the complicated regulation mechanisms and some novel functions of CBL-CIPK signaling network in plants need to be further elucidated, numerous advances have been made in its roles involved in the abiotic stresses. This review chiefly introduces the progresses about protein interaction, classification and expression pattern of different CBLs and CIPKs in Arabidopsis thaliana, summarizes the physiological roles of CBL-CIPK pathway while pointing out some new research ideas in the future, and finally presents some unique perspectives for the further study. The review might provide new insights into the functional characterization of CBL-CIPK pathway in Arabidopsis, and contribute to a deeper understanding of CBL-CIPK network in other plants or stresses.

Genome-wide identification and expression analysis of the WRKY gene family in common tobacco (Nicotiana tabacum L.).

The coding products of WRKY gene family plays important roles in plant growth and development as well as in various stress responses. They have been identified in various plants, but only few in common tobacco (Nicotiana tabacum L.). In this study, 164 putative WRKY proteins in the common tobacco genome were identified by using the conserved WRKY sequence (PF03106) from the Pfam database. Phylogenetic trees, functional domain analysis, chromosomal localization, subcellular localization and tissue expression patterns were analyzed with the bioinformatics softwares, including DNAMAN 5.0, Weblogo 3, MEGA 5.1, MG2C and MEME. First of all, phylogenetic trees divided all the candidate genes into three subfamilies: Ⅰ, Ⅱ and Ⅲ, respectively, and subfamily Ⅱ could be further divided into five subgroups: group Ⅱ-a, -b, -c, -d and -e. Secondly, the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK followed by a zinc-finger motif. Most of the NtWRKY genes contained 2-5 exons and a highly conserved gene structure. Thirdly, 154 out of 164 NtWRKY genes were distributed with different densities on 24 chromosomes, and each subfamily with different patterns and frequency. The largest number of NtWRKY genes was found on chromosome VI, and only one on chromosome X. Fourthly, the majority of NtWRKY members located in the nucleus, with 74 percent of subfamily Ⅲ in the extracellular matrix. Lastly, the members in the same subfamily had different spatial and temporal expression profiles, with 11 NtWRKY genes in roots, stems and leaves expressed at various levels. The expression of genes NtWRKY26, NtWRKY30 and NtWRKY32 can be induced by Phytophthora nicotianae. Our research thus provides valuable information for NtWRKY gene cloning and functional characterization in common tobacco.

Genome-wide identification and expression profiling of the C2H2-type zinc finger protein transcription factor family in tobacco.

C2H2 zinc finger protein transcription factor family members have important biological functions in eukaryotes. They not only bind DNA and RNA, but also interact with proteins. In this study, 118 members of the tobacco C2H2 zinc finger protein transcription factor family were identified from the N. tabacum genome database by using Pfam, SMART and Blastp. The analyses of phylogenetic tree, physical and chemical properties, chromosomal mapping, gene structures, protein three-dimensional structures and tissue expression patterns were performed. The results suggested that the peptide length of different subfamily members is significantly different. Phylogenetic and motif analysis revealed that the C2H2 zinc finger protein transcription factor family members can be divided into 5 subfamilies and each member has at least one C2H2 motif. Genes of the family members are distributed across the 22 chromosomes. C2H2 zinc finger protein transcription factor family members are expressed in different tissues although some have higher expression levels in leaves and roots. This study will be helpful for further analysis of the C2H2 zinc finger family proteins in other plants.

MapGene2Chrom, a tool to draw gene physical map based on Perl and SVG languages.

Genetic linkage map is helpful for analysis on heredity of some gene families and map-based gene cloning because of its simple and elegant manifestation. One software is in need to draw a gene physical map, which shows a manner similar to the genetic linkage map, based on the relative physical distance between genes. Although some tools like GBrowse and MapViewer etc. are available to draw gene physical map, there are obvious limitations for them: (1) the data need to be decorated in advance; (2) users can't modify results. Therefore, we developed a bio-assisted mapping software--MapGene2Chrom with PC and web versions, which is based on Perl and SVG languages. The software can be used to draw the corresponding physical map quickly in SVG format based on the input data. It will become a useful tool for drawing gene physical map with the advantages of simple input data format, easily modified output and very good portability.

[Bioinformatic prediction of conserved microRNAs and their target genes in eggplant (Solanum melongena L.)].

MicroRNAs (miRNAs), a recently discovered class of small (-21nt), non-coding, endogenous, single-stranded RNAs in eukaryotes, regulate gene expression negatively at the post-transcriptional levels depending on the extent of complementation between miRNA and mRNA. To date, a large number of miRNAs have been reported in many species, but none for eggplant (Solanum melongena L.). In this paper, a computational homology search approach based on the conservation of miRNA sequences and the stem-loop hairpin secondary structures of miRNAs was adopted. The search was started with the known plant miRNAs compared to eggplant expressed sequence tags (EST) databases to find potential miRNAs. Following a range of filtering criteria, a total of 16 potential miRNAs belonging to 12 families were identified. Three pairs of sense and antisense strand eggplant miRNAs belonging to three different miRNA families were also found. Furthermore, miR390 and miR399 sense/antisense pairs are identified for the first time in plants. Using online software psRNATarget, we further predicted the target genes of these 16 miRNAs and got 71 potential targets genes on base of 15 eggplant miRNAs. Most of these target genes were predicted to encode proteins that play key role in eggplant growth, development, metabolism, and stress responses.