Cloning of cDNA encoding the bifunctional dehydroquinase.shikimate dehydrogenase of aromatic-amino-acid biosynthesis in Nicotiana tabacum.
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ABSTRACT:
SUBMITTER: Bonner CA
PROVIDER: S-EPMC1137184 | biostudies-other | 1994 Aug
REPOSITORIES: biostudies-other
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Project description:Polygalacturonase inhibiting proteins (PGIPs) are major defensive proteins produced by plant cell walls that play a crucial role in pathogen resistance by reducing polygalacturonase (PG) activity. In the present study, a novel PGIP gene was isolated from tobacco (Nicotiana tabacum), hereafter referred as NtPGIP. A full-length NtPGIP cDNA of 1,412 bp with a 186 bp 5'-untranslated region (UTR), and 209 bp 3'-UTR was cloned from tobacco, NtPGIP is predicted to encode a protein of 338 amino acids. The NtPGIP sequence from genomic DNA showed no introns and sequence alignments of NtPGIP's deduced amino acid sequence showed high homology with known PGIPs from other plant species. Moreover, the putative NtPGIP protein was closely clustered with several Solanaceae PGIPs. Further, the expression profile of NtPGIP was examined in tobacco leaves following stimulation with the oomycete Phytophthora nicotianae and other stressors, including salicylic acid (SA), abscisic acid (ABA), salt, and cold treatment. The results showed that all of the treatments up-regulated the expression of NtPGIP at different times. To understand the biochemical activity of NtPGIP gene, a full-length NtPGIP cDNA sequence was subcloned into a pET28a vector and transformed into E. coli BL21 (DE3). Recombinant proteins were successfully induced by 1.0 nmol/L IPTG and the purified proteins effectively inhibited Phytophthora capsici PG activity. The results of this study suggest that NtPGIP may be a new candidate gene with properties that could be exploited in plant breeding.
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Project description:As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis as well as photoprotectors for plants to adapt to high light stress. Phytoene synthase (PSY) is the entry enzyme and also the major rate-limiting enzyme in the carotenoid pathway. Here, we report a dehydration-responsive element-binding protein (DREB) transcription factor member in Nicotiana tabacum K326, NtDREB-1BL1, which regulates carotenoids biosynthesis by binding to the NtPSY promoter. The NtDREB-1BL1 transcript was widely distributed in leaves by Real-time PCR. Confocal image revealed that NtDREB-1BL1 was localized in the nucleus. The chromatin immunoprecipitation (ChIP) with the qPCR technique indicated that NtDREB-1BL1 could anchor the promoter region of NtPSY. Overexpression (NtDREB-1BL1 OE) and RNA interference (NtDREB-1BL1 RNAi) of NtDREB-1BL1 were performed to evaluate its biological function in N. tabacum. Both carotenoid and chlorophyll contents increased in transgenic plants of NtDREB-1BL1 OE compared with wild-type (WT) plants, with the augment of the genes involved in carotenoid biosynthesis. In contrast, the contents of carotenoid and chlorophyll significantly decreased in transgenic plants of NtDREB-1BL1 RNAi compared to WT, along with the decline in the expression of genes related to carotenoid biosynthesis. Moreover, transgenic plants of NtDREB-1BL1 OE exhibited enhanced tolerance under drought stress, with the weakened tolerance of drought stress in transgenic plants of NtDREB-1BL1 RNAi. In conclusion, our results illustrated the new role of transcription factor NtDREB-1BL1 in improving carotenoid biosynthesis through regulating NtPSY expression.
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Project description:Wheat is recalcitrant to genetic transformation. A potential solution is to manipulate the expression of some host proteins involved in T-DNA integration process. VirE2 interacting protein 2 (VIP2) plays an important role in T-DNA transport and integration. In this study, a TaVIP2 gene was cloned from common wheat. Southern blot and allele-specific polymerase chain reaction (AS-PCR) combined with an online chromosomal location software tool revealed that three TaVIP2 genes were located on wheat chromosomes 1AL, 1BL, and 1DL. These three homoeoallelic TaVIP2 genes all contained 13 exons and 12 introns, and their coding sequences were the same; there were a few single nucleotide polymorphisms (SNPs) among the three genes. The heterologous expression of the TaVIP2 gene in tobacco led to enhancement of the Agrobacterium-mediated transformation efficiency up to 2.5-fold. Transgenic tobacco plants expressing TaVIP2 showed enhanced resistance to powdery mildew. Further quantitative real-time PCR (qRT-PCR) revealed that overexpression of TaVIP2 in transgenic tobacco up-regulated the expression of an endogenous gene, NtPR-1, which likely contributed to powdery mildew resistance in transgenic tobacco. Our study indicates that the TaVIP2 gene may be highly useful in efforts to improve Agrobacterium-mediated transformation efficiency and to enhance powdery mildew resistance in wheat.
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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:Because of their special organization, multifunctional enzymes play crucial roles in improving the performance of metabolic pathways. For example, the bacterium Prevotella nigrescens contains a distinctive bifunctional protein comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS), catalyzing the first reaction of the biosynthetic pathway of aromatic amino acids, and a chorismate mutase (CM), functioning at a branch of this pathway leading to the synthesis of tyrosine and phenylalanine. In this study, we characterized this P. nigrescens enzyme and found that its two catalytic activities exhibit substantial hetero-interdependence and that the separation of its two distinct catalytic domains results in a dramatic loss of both DAH7PS and CM activities. The protein displayed a unique dimeric assembly, with dimerization solely via the CM domain. Small angle X-ray scattering (SAXS)-based structural analysis of this protein indicated a DAH7PS-CM hetero-interaction between the DAH7PS and CM domains, unlike the homo-association between DAH7PS domains normally observed for other DAH7PS proteins. This hetero-interaction provides a structural basis for the functional interdependence between the two domains observed here. Moreover, we observed that DAH7PS is allosterically inhibited by prephenate, the product of the CM-catalyzed reaction. This allostery was accompanied by a striking conformational change as observed by SAXS, implying that altering the hetero-domain interaction underpins the allosteric inhibition. We conclude that for this C-terminal CM-linked DAH7PS, catalytic function and allosteric regulation appear to be delivered by a common mechanism, revealing a distinct and efficient evolutionary strategy to utilize the functional advantages of a bifunctional enzyme.
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Project description:UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.
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Project description:Phosphorus (P) is an essential macronutrient for plant growth and development. The concentration of flavonol, a natural plant antioxidant, is closely related to phosphorus nutritional status. However, the regulatory networks of flavonol biosynthesis under low Pi stress are still unclear. In this study, we identified a PFG-type MYB gene, NtMYB12, whose expression was significantly up-regulated under low Pi conditions. Overexpression of NtMYB12 dramatically increased flavonol concentration and the expression of certain flavonol biosynthetic genes (NtCHS, NtCHI, and NtFLS) in transgenic tobacco. Moreover, overexpression of NtMYB12 also increased the total P concentration and enhanced tobacco tolerance of low Pi stress by increasing the expression of Pht1-family genes (NtPT1 and NtPT2). We further demonstrated that NtCHS-overexpressing plants and NtPT2-overexpressing plants also had increased flavonol and P accumulation and higher tolerance to low Pi stress, showing a similar phenotype to NtMYB12-overexpressing transgenic tobacco under low Pi stress. These results suggested that tobacco NtMYB12 acts as a phosphorus starvation response enhancement factor and regulates NtCHS and NtPT2 expression, which results in increased flavonol and P accumulation and enhances tolerance to low Pi stress.
| S-EPMC6993060 | biostudies-literature
Project description:BackgroundTobacco (Nicotiana tabacum) is an important plant model system that has played a key role in the early development of molecular plant biology. The tobacco genome is large and its characterisation challenging because it is an allotetraploid, likely arising from hybridisation between diploid N. sylvestris and N. tomentosiformis ancestors. A draft assembly was recently published for N. tabacum, but because of the aforementioned genome complexities it was of limited utility due to a high level of fragmentation.ResultsHere we report an improved tobacco genome assembly, which, aided by the application of optical mapping, achieves an N50 size of 2.17 Mb and enables anchoring of 64% of the genome to pseudomolecules; a significant increase from the previous value of 19%. We use this assembly to identify two homeologous genes that explain the differentiation of the burley tobacco market class, with potential for greater understanding of Nitrogen Utilization Efficiency and Nitrogen Use Efficiency in plants; an important trait for future sustainability of agricultural production.ConclusionsDevelopment of an improved genome assembly for N. tabacum enables what we believe to be the first successful map-based gene discovery for the species, and demonstrates the value of an improved assembly for future research in this model and commercially-important species.
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Project description:Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.
| S-EPMC4691183 | biostudies-literature
Project description:Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco.
| S-EPMC312006 | biostudies-other