A gene signature linked to fibroblast differentiation for prognostic prediction of mesothelioma.

BACKGROUND: Malignant mesothelioma is a type of infrequent tumor that is substantially related to asbestos exposure and has a terrible prognosis. We tried to produce a fibroblast differentiation-related gene set for creating a novel classification and prognostic prediction model of MESO. METHOD: Three databases, including NCBI-GEO, TCGA, and MET-500, separately provide single-cell RNA sequencing data, bulk RNA sequencing profiles of MESO, and RNA sequencing information on bone metastatic tumors. Dimensionality reduction and clustering analysis were leveraged to acquire fibroblast subtypes in the MESO microenvironment. The fibroblast differentiation-related genes (FDGs), which were associated with survival and subsequently utilized to generate the MESO categorization and prognostic prediction model, were selected in combination with pseudotime analysis and survival information from the TCGA database. Then, regulatory network was constructed for each MESO subtype, and candidate inhibitors were predicted. Clinical specimens were collected for further validation. RESULT: A total of six fibroblast subtypes, three differentiation states, and 39 FDGs were identified. Based on the expression level of FDGs, three MESO subtypes were distinguished in the fibroblast differentiation-based classification (FDBC). In the multivariate prognostic prediction model, the risk score that was dependent on the expression level of several important FDGs, was verified to be an independently effective prognostic factor and worked well in internal cohorts. Finally, we predicted 24 potential drugs for the treatment of MESO. Moreover, immunohistochemical staining and statistical analysis provided further validation. CONCLUSION: Fibroblast differentiation-related genes (FDGs), especially those in low-differentiation states, might participate in the proliferation and invasion of MESO. Hopefully, the raised clinical subtyping of MESO would provide references for clinical practitioners.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis.

BACKGROUND: UDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides. RESULTS: In this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark. CONCLUSION: Our findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

A two-step mutation process in the double WS1 homologs drives the evolution of burley tobacco, a special chlorophyll-deficient mutant with abnormal chloroplast development.

MAIN CONCLUSION: The functional homologs WS1A and WS1B, identified by map-based cloning, control the burley character by affecting chloroplast development in tobacco, contributing to gene isolation and genetic improvement in polyploid crops. Burley represents a special type of tobacco (Nicotiana tabacum L.) cultivar that is characterized by a white stem with a high degree of chlorophyll deficiency. Although important progress in the research of burley tobacco has been made, the molecular mechanisms underlying this character remain unclear. Here, on the basis of our previous genetic analyses and preliminary mapping results, we isolated the White Stem 1A (WS1A) and WS1B genes using a map-based cloning approach. WS1A and WS1B are functional homologs with completely identical biological functions and highly similar expression patterns that control the burley character in tobacco. WS1A and WS1B are derived from Nicotiana sylvestris and Nicotiana tomentosiformis, the diploid ancestors of Nicotiana tabacum, respectively. The two genes encode zinc metalloproteases of the M50 family that are highly homologous to the Ethylene-dependent Gravitropism-deficient and Yellow-green 1 (EGY1) protein of Arabidopsis and the Lutescent 2 (L2) protein of tomato. Transmission electron microscopic examinations indicated that WS1A and WS1B are involved in the development of chloroplasts by controlling the formation of thylakoid membranes, very similar to that observed for EGY1 and L2. The genotyping of historical tobacco varieties revealed that a two-step mutation process occurred in WS1A and WS1B during the evolution of burley tobacco. We also discussed the strategy for gene map-based cloning in polyploid plants with complex genomes. This study will facilitate the identification of agronomically important genes in tobacco and other polyploid crops and provide insights into crop improvement via molecular approaches.

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.

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.

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.