De novo transcriptome assembly of transgenic tobacco (Nicotiana tabacum NC89) with early senescence characteristic.
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ABSTRACT: Supplementary information
The online version contains supplementary material available at 10.1007/s12298-021-00953-z.
SUBMITTER: Liu H
PROVIDER: S-EPMC7907299 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Backgroundα-Farnesene is a volatile sesquiterpene synthesized by the plant mevalonate (MVA) pathway through the action of α-farnesene synthase. The α-farnesene synthase 1 (MdAFS1) gene was isolated from apple peel (var. white winter pearmain), and transformed into tobacco (Nicotiana tabacum NC89). The transgenic plants had faster stem elongation during vegetative growth and earlier flowering than wild type (WT). Our studies focused on the transgenic tobacco phenotype.ResultsThe levels of chlorophyll and soluble protein decreased and a lower seed biomass and reduced net photosynthetic rate (Pn) in transgenic plants. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide radicals (O 2 (·-) ) had higher levels in transgenics compared to controls. Transgenic plants also had enhanced sensitivity to oxidative stress. The transcriptome of 8-week-old plants was studied to detect molecular changes. Differentially expressed unigene analysis showed that ubiquitin-mediated proteolysis, cell growth, and death unigenes were upregulated. Unigenes related to photosynthesis, antioxidant activity, and nitrogen metabolism were downregulated. Combined with the expression analysis of senescence marker genes, these results indicate that senescence started in the leaves of the transgenic plants at the vegetative growth stage.ConclusionsThe antioxidative defense system was compromised and the accumulation of reactive oxygen species (ROS) played an important role in the premature aging of transgenic plants.
| S-EPMC4930573 | biostudies-literature
Project description:Senescence is a degenerative process triggered by intricate and coordinated regulatory networks, and the mechanisms of age-dependent senescence and stress-induced premature senescence still remain largely elusive. Thus we selected leaf samples of developmental senescence (DS) and premature senescence (PS) to reveal the regulatory divergence. Senescent leaves were confirmed by yellowing symptom and physiological measurement. A total of 1171 and 309 genes (DEGs) were significantly expressed respectively in the whole process of DS and PS. Up-regulated DEGs in PS were mostly related to ion transport, while the down-regulated DEGs were mainly associated with oxidoreductase activity and sesquiterpenoid and triterpenoid biosynthesis. In DS, photosynthesis, precursor metabolites and energy, protein processing in endoplasmic reticulum, flavonoid biosynthesis were notable. Moreover, we found the vital pathways shared by DS and PS, of which the DEGs were analyzed further via protein-protein interaction (PPI) network analysis to explore the alteration responding to two types of senescence. In addition, plant hormone transduction pathway was mapped by related DEGs, suggesting that ABA and ethylene signaling played pivotal roles in formulating the distinction of DS and PS. Finally, we conducted a model containing oxidative stress and ABA signaling as two hub points, which highlighted the major difference and predicted the possible mechanism under DS and PS. This work gained new insight into molecular divergence of developmental senescence and premature senescence and would provide reference on potential mechanism initiating and motivating senescence for further study.
| S-EPMC7688636 | biostudies-literature
Project description:BackgroundPollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. Cellular and regulatory mechanisms underlying pollen tube elongation are under intense investigation, which stands to greatly benefit from a comprehensive understanding of global gene expression profiles in pollen and pollen tubes. Here, RNA sequencing technology was applied to de novo assemble a Nicotiana tabacum male gametophytic transcriptome and to compare transcriptome profiles at two different stages of gametophyte development: mature pollen grains (MPG) and pollen tubes grown for six hours in vitro (PT6).ResultsDe novo assembly of data obtained by 454 sequencing of a normalized cDNA library representing tobacco pollen and pollen tube mRNA (pooled mRNA isolated from mature pollen grains [MPG] and from pollen tubes grown in vitro for 3 [PT3] or 6 [PT6] hours) resulted in the identification of 78,364 unigenes. Among these unigenes, which mapped to 24,933 entries in the Sol Genomics Network (SGN) N. tabacum unigene database, 24,672 were predicted to represent full length cDNAs. In addition, quantitative analyses of data obtained by Illumina sequencing of two separate non-normalized MPG and PT6 cDNA libraries showed that 8979 unigenes were differentially expressed (differentially expressed unigenes: DEGs) between these two developmental stages at a FDR q-value of <0.0001. Interestingly, whereas most of these DEGs were downregulated in PT6, the minor fraction of DEGs upregulated in PT6 was enriched for GO (gene ontology) functions in pollen tube growth or fertilization.ConclusionsA major output of our study is the development of two different high-quality databases representing the tobacco male gametophytic transcriptome and containing encompassing information about global changes in gene expression after pollen germination. Quantitative analyses of these databases 1) indicated that roughly 30% of all tobacco genes are expressed in the male gametophyte, and 2) support previous observations suggesting a global reduction of transcription after pollen germination. Interestingly, a small number of genes, many of which predicted to function in pollen tube growth or fertilization, were found to be upregulated in elongating pollen tubes despite globally reduced transcription.
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Project description:1. Coproporphyrinogenase was extracted and purified from tobacco (Nicotiana tabacum L.). Enzyme activity was mainly located in mitochondria rather than in chloroplasts. The enzyme was purified by differential centrifugation, ammonium sulphate fractionation, calcium phosphate gel adsorption and dialysis. A 69-fold final purification was obtained. 2. An apparent K(m) value of 3.6x10(-5)m was found, the value being largely dependent on the amount of coproporphyrin III recovered after reduction with sodium amalgam to coproporphyrinogen III. Protoporphyrin formation was linear up to 3h and decreased with further incubation. The enzyme activity increased with the concentration of enzyme protein up to 30mug/ml of solution. 3. Enzyme activity was greatly enhanced by increasing Fe(2+) concentrations up to 0.5mm, beyond which inhibition occurred. Co(2+) and Mn(2+) were also found to activate at low concentrations (0.1mm) and inhibit at higher concentrations (5mm). Fe(3+) and Cu(2+), both at 0.1mm, and o-phenanthroline and EDTA, each at 1mm, were found to be inhibitory.
| S-EPMC1178852 | biostudies-other
Project description:BACKGROUND:Nicotiana benthamiana has been widely used in laboratories around the world for studying plant-pathogen interactions and posttranscriptional gene expression silencing. Yet the exploration of its transcriptome has lagged behind due to the lack of both adequate sequence information and genome-wide analysis tools, such as DNA microarrays. Despite the increasing use of high-throughput sequencing technologies, the DNA microarrays still remain a popular gene expression tool, because they are cheaper and less demanding regarding bioinformatics skills and computational effort. RESULTS:We designed a gene expression microarray with 103,747 60-mer probes, based on two recently published versions of N. benthamiana transcriptome (v.3 and v.5). Both versions were reconstructed from RNA-Seq data of non-strand-specific pooled-tissue libraries, so we defined the sense strand of the contigs prior to designing the probe. To accomplish this, we combined a homology search against Arabidopsis thaliana proteins and hybridization to a test 244k microarray containing pairs of probes, which represented individual contigs. We identified the sense strand in 106,684 transcriptome contigs and used this information to design an Nb-105k microarray on an Agilent eArray platform. Following hybridization of RNA samples from N. benthamiana roots and leaves we demonstrated that the new microarray had high specificity and sensitivity for detection of differentially expressed transcripts. We also showed that the data generated with the Nb-105k microarray may be used to identify incorrectly assembled contigs in the v.5 transcriptome, by detecting inconsistency in the gene expression profiles, which is indicated using multiple microarray probes that match the same v.5 primary transcripts. CONCLUSIONS:We provided a complete design of an oligonucleotide microarray that may be applied to the research of N. benthamiana transcriptome. This, in turn, will allow the N. benthamiana research community to take full advantage of microarray capabilities for studying gene expression in this plant. Additionally, by defining the sense orientation of over 106,000 contigs, we substantially improved the functional information on the N. benthamiana transcriptome. The simple hybridization-based approach for detecting the sense orientation of computationally assembled sequences can be used for updating the transcriptomes of other non-model organisms, including cases where no significant homology to known proteins exists.
| S-EPMC4875705 | biostudies-literature
Project description:BackgroundNicotiana benthamiana has been widely used for transient gene expression assays and as a model plant in the study of plant-microbe interactions, lipid engineering and RNA silencing pathways. Assembling the sequence of its transcriptome provides information that, in conjunction with the genome sequence, will facilitate gaining insight into the plant's capacity for high-level transient transgene expression, generation of mobile gene silencing signals, and hyper-susceptibility to viral infection.Methodology/resultsRNA-seq libraries from 9 different tissues were deep sequenced and assembled, de novo, into a representation of the transcriptome. The assembly, of 16GB of sequence, yielded 237,340 contigs, clustering into 119,014 transcripts (unigenes). Between 80 and 85% of reads from all tissues could be mapped back to the full transcriptome. Approximately 63% of the unigenes exhibited a match to the Solgenomics tomato predicted proteins database. Approximately 94% of the Solgenomics N. benthamiana unigene set (16,024 sequences) matched our unigene set (119,014 sequences). Using homology searches we identified 31 homologues that are involved in RNAi-associated pathways in Arabidopsis thaliana, and show that they possess the domains characteristic of these proteins. Of these genes, the RNA dependent RNA polymerase gene, Rdr1, is transcribed but has a 72 nt insertion in exon1 that would cause premature termination of translation. Dicer-like 3 (DCL3) appears to lack both the DEAD helicase motif and second dsRNA binding motif, and DCL2 and AGO4b have unexpectedly high levels of transcription.ConclusionsThe assembled and annotated representation of the transcriptome and list of RNAi-associated sequences are accessible at www.benthgenome.com alongside a draft genome assembly. These genomic resources will be very useful for further study of the developmental, metabolic and defense pathways of N. benthamiana and in understanding the mechanisms behind the features which have made it such a well-used model plant.
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Project description:Leaf senescence is an important process of growth and development in plant, and it is a programmed decline controlled by a series of genes. In this study, the biochemical properties and transcriptome at five maturity stages (M1∼M5) of tobacco leaves were analyzed to reveal the dynamic changes in leaf senescence of tobacco. A total of 722, 1,534, 3,723, and 6,933 genes were differentially expressed (DEG) between M1 and M2, M1 and M3, M1 and M4, and M1 and M5, respectively. Significant changes of nitrogen, sugars, and the DEGs related to metabolite accumulation were identified, suggesting the importance of energy metabolism during leaf senescence. Gene Ontology (GO) analysis found that DEGs were enriched in biosynthetic, metabolic, photosynthesis, and redox processes, and especially, the nitrogen metabolic pathways were closely related to the whole leaf senescence process (M1∼M5). All the DEGs were grouped into 12 expression profiles according to their distinct expression patterns based on Short Time-series Expression Miner (STEM) software analysis. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that these DEGs were enriched in pathways of carbon metabolism, starch and sucrose metabolism, nitrogen metabolism, and photosynthesis among these expression profiles. A total of 30 core genes were examined by Weight Gene Co-expression Network Analysis (WGCNA), and they appeared to play a crucial role in the regulatory of tobacco senescence. Our results provided valuable information for further functional investigation of leaf senescence in plants.
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Project description:Seed-specific expression using appropriate promoters is a recommended strategy for the efficiently producing valuable metabolites in transgenic plants. In the present study, we investigated the sequence of sucrose binding protein (SBP) as a seed-specific promoter to find the cis-acting elements specific to gene expression in seeds. The 1860 bp SBP sequence was analyzed using Plant Care and PLACE databases to find cis-acting elements, which resulted in a finding of 22 cis-acting elements required for seed expression. In addition, we have discovered cis- acting elements that are indirectly involved in triacylglycerol synthesis (GATABOX, DOFCOREZM, CACGTGMOTIF). The seed specificity of SBP was analyzed by generating a stable transgenic tobacco plant harboring β-glucuronidase (GUS) reporter gene under the control of the SBP promoter. Histochemical analysis of these transgenic tobacco plants indicated decreasing GUS activity in the leaves during the vegetative stage. However, the mature seeds of transgenic plants showed GUS activity. Moreover, the SBP promoter function in the seed oil content was evaluated by the expression of DGAT1. The expression analysis of DGAT1 in SBP-DGAT1 transgenic tobacco seeds using quantitative real-time PCR revealed a 7.8-fold increase in DGAT1 than in non-transgenic plants. Moreover, oil content increased up to 2.19 times more than in non-transgenic plants. And the oil content of the SBP-DGAT1 transgenic tobacco leaves did not change compared to the control plant. Therefore, we suggested that the SBP promoter could be used as a seed-specific promoter for targeted expression of desired genes in the metabolite engineering of oilseed crops.
| S-EPMC9165846 | biostudies-literature
Project description:Senescence affects the remobilization of nutrients and adaption of the plant to the environment. Combined stresses can result in premature senescence in plants which exist in the field. In this study, transcriptomic analysis was performed on mature leaves and leaves in three stages of premature senescence to understand the molecular mechanism. With progressive premature senescence, a declining chlorophyll (chl) content and an increasing malonaldehyde (MDA) content were observed, while plasmolysis and cell nucleus pyknosis occurred, mitochondria melted, thylakoid lamellae were dilated, starch grains in chloroplast decreased, and osmiophilic granules increased gradually. Moreover, in total 69 common differentially expressed genes (DEGs) in three stages of premature senescing leaves were found, which were significantly enriched in summarized Gene Ontology (GO) terms of membrane-bounded organelle, regulation of cellular component synthesis and metabolic and biosynthetic processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the plant hormone signal transduction pathway was significantly enriched. The common DEGs and four senescence-related pathways, including plant hormone signal transduction, porphyrin and chlorophyll metabolism, carotenoid biosynthesis, and regulation of autophagy were selected to be discussed further. This work aimed to provide potential genes signaling and modulating premature senescence as well as the possible dynamic network of gene expression patterns for further study.
| S-EPMC6278766 | biostudies-literature
Project description:Gene expression microarray was designed, based on two recently published versions of the N. benthamiana transcriptome (v.3 and v.5). Based on homology searches and hybridization tests we identified the sense strand in 106,684 transcriptome contigs and used this information to design a Nb-105k microarray on Agilent eArray platform. By the hybridization of N. benthamiana roots vs leaves RNA samples we showed high specificity and sensitivity of the new microarray in detection of differentially expressed transcripts. Agilent microarrays in 2x105k format (two microarrays per slide) were used for hybridization of labeled RNA from leaves vs roots of N. benthamiana plants. Four biological replicates were used for two-color design in a dye-swap manner.
2016-01-08 | E-GEOD-76631 | biostudies-arrayexpress