Nicotiana Alata Link Research Articles

Prediction of global potential suitable habitats of Nicotiana alata Link et Otto based on MaxEnt model

Nicotiana alata Link et Otto, widely used in landscaping, is not only of great ornamental value but also of high commercial and medical value. The global potential habitat of N. alata and the environmental factors affecting its distribution are not that clear at present. To provide a reference for the reasonable and extensive planting of N. alata now and in the future, the MaxEnt model was used to predict its global suitable habitats under current and future climate conditions, respectively, based on global geographic distribution data of N. alata and the current and future world bioclimatic variables. The results showed that mean temperature of the driest quarter (bio9), precipitation of driest month (bio14), precipitation seasonality (bio15) and max temperature of warmest month (bio5), were the key bioclimatic variables governing the distribution of N. alata. The global suitable habitats of N. alata were mainly distributed in Europe, the United States, southeastern South America, and China under current climate conditions. Compared with current climate conditions, the future climate decreased suitable habitats of N. alata under SSP1-2.6, and SSP2-4.5 scenario and increased suitable habitats of N. alata under SSP3-7.0, and SSP5-8.5 climatic scenarios. The results provided valuable information and theoretical reference for the reasonable planting of N. alata.

Report of Tetranychus evansi Baker & Pritchard (Acari: Tetranychidae) in species of the genus Nicotiana (Solanaceae)

Tetranychus evansi Baker & Pritchard, 1960 (Tetranychidae) is a phytophagous mite with wide distribution and high importance for agriculture. This study reports the occurrence of T. evansi in Nicotiana alata Link & Otto, Nicotiana forgetiana Sander ex W. Watson (Solanaceae) and their putative hybrids in a greenhouse in Rio Grande do Sul state, Brazil. Different stages of development of T. evansi were found in N. alata and N. forgetiana and in their putative hybrids.

Effects of different potting growing media for Petunia grandiflora and Nicotiana alata Link & Otto on photosynthetic capacity, leaf area, and flowering potential

Petunia grandiflora Juss. and Nicotiana alata Link & Otto are two of the most widely spread plants on the market for annual potted ornamental plants. In order to identify the most adequate substrate formula we analyzed the effects of different potting growing media used for P. hybrida grandiflora 'Bravo' and N. alata 'Dinamo' on their photosynthetic capacity, leaf area, and flowering potential. Optimization of growing media formula for petunia and ornamental tobacco was performed by preparing four growing media mixing fallow soil (FS), Biolan peat (BP), acid peat (AP), leaf compost (C), and perlite (P) in different proportions. The physiological potential of petunia and ornamental tobacco was investigated by photosynthesis and respiration rate and chlorophyll pigments in leaves, while the vegetative and flowering phenological stages were evaluated by number of leaves per plant, leaf area, number of flowers per plant and leaf area/flowers ratio. These measurements were significantly influenced by the different potting growing media used in this study. In the flowering stage, the highest photosynthesis rates (8.612 μmol CO2 m-2 s-1) as well as leaf area (1.766 dm2) of petunias were obtained on growing media with 60% biolan peat, 30% acid peat and 10% perlite (BP60-AP30-P10). Flowering responses to growing conditions vary greatly among plants and the biggest number of ornamental tobacco flowers (22 flowers plant-1) was registered as an effect of BP60-AP30-P10 media. Growing media with the BP60-AP30-P10 formula seem to be the most adequate growth substrate to develop profitable crops for petunias and ornamental tobacco with high decorative value.

Pest and disease protection conferred by expression of barley β-hordothionin and Nicotiana alata proteinase inhibitor genes in transgenic tobacco.

Proteinase inhibitors and thionins are among the many proteins thought to have a role in plant defence against pests and pathogens. Complementary DNA clones encoding the precursors of a multi-domain proteinase inhibitor from Nicotiana alata Link et Otto (NA-PI) (Mr approximately 43 000) and a β-hordothionin (β-HTH) (Mr approximately 13 000) from barley, were linked to constitutive promoters and subsequently transferred by Agrobacterium-mediated transformation into tobacco. The NA-PI and β-HTH precursor proteins were synthesised and post-translationally processed in transgenic tobacco and accumulated as polypeptides of apparent size Mr approximately 6000 and Mr approximately 8500, respectively. The na-pi and β-hth genes were stably inherited for at least two generations. Transgenic tobacco plants containing the highest amounts of NA-PI and β-HTH were crossed to produce plants containing both genes. Helicoverpa armigera (tobacco budworm) larvae that ingested transgenic tobacco leaves expressing both NA-PI and β-HTH, exhibited higher mortality and slower development relative to larvae fed on non-transgenic tobacco. NA-PI and β-HTH, either alone, or in combination, also conferred protection against the fungal pathogen, Botrytis cinerea (grey mould) and the bacterial pathogen, Pseudomonas solanacearum (bacterial wilt). The effect of the two proteins depended upon the organism tested and the contribution of each gene to the protective effects was not necessarily equal. The genetic engineering of plants with proteinase inhibitors or thionins, therefore, has potential for improving crop productivity by simultaneously increasing resistance to both pests and pathogens.

Analysis of wound-induced gene expression in Nicotiana species with contrasting alkaloid profiles.

We determined the capacity of three Nicotiana (Solanaceae) species with very different alkaloid profiles (Nicotiana sylvestris Speg & Comes, Nicotiana alata Link & Otto and Nicotiana glauca Grah.) to increase their alkaloid contents in both leaf and root tissues following foliage damage. We also investigated the transcriptional responses of genes encoding enzymes important for alkaloid biosynthesis, namely quinolinate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC) and the putative alkaloid biosynthetic gene A622. In response to wounding of foliage in the well studied 'model' species N. sylvestris, a rise, approximately 2-fold, in leaf nicotine levels was observed several days after a 4-5-fold increase in the transcript levels of all genes in the roots. In contrast, leaf tissues of the ornamental tobacco N. alata showed very low levels of any pyridine alkaloid, even when analysed 1 week after wounding, correlating with a general lack of transcript abundance representing any of these genes in leaves or roots following foliage damage. However, addition of methyl jasmonate to cultured roots of N. alata did produce elevated levels of nicotine and anatabine raising the possibility that components of the leaf-root wound signalling system in N. alata are different from those in N. sylvestris. Wounding of the tree tobacco N. glauca, was followed by a 2-fold increase in anabasine levels several days later. This increase followed a large rise in transcript levels of ODC, QPT and A622, though not PMT, in wounded leaves, but not in non-wounded leaves or roots. These data support the hypothesis that N. glauca is able to produce increased anabasine levels following wounding in its foliage, setting it apart from N. sylvestris where induced alkaloid production takes place in roots. We discuss the possibility that increased transcript levels detected by ODC and A622 probes play important roles in anabasine synthesis in N. glauca.

Response of Nicotiana alata to Insertion of an Autoregulated Senescence- inhibition Gene

Nicotiana alata Link and Otto (Jasmine tobacco) was transformed with an autoregulated senescence-inhibition gene construct PSAG12-IPT encoding isopentenyl transferase via Agrobacterium-mediated transformation. Transformation was confirmed by polymerase chain reaction. Transgenic plants exhibited up to 2- to 4-fold fewer senesced leaves, 29% longer in situ flower life, 26% more shoot dry weight, and a 32% to 50% reduction in flowers per branch. Additionally, transgenics were 28% shorter and had up to 174% more branches, indicative of cytokinin overproduction and a lack of tight autoregulation of PSAG12-IPT. Variation among independent transgenics suggests selection for enhanced PSAG12-IPT is feasible. Our observations of increased branching and in situ flower longevity, as well as reduced plant height and flowers per branch provide new information on PSAG12-IPT and its potential value for biological study and horticultural application.

Pollen tubes of Nicotiana alata express two genes from different beta-glucan synthase families.

The walls deposited by growing pollen tubes contain two types of beta-glucan, the (1,3)-beta-glucan callose and the (1,4)-beta-glucan cellulose, as well as various alpha-linked pectic polysaccharides. Pollen tubes of Nicotiana alata Link et Otto, an ornamental tobacco, were therefore used to identify genes potentially encoding catalytic subunits of the callose synthase and cellulose synthase enzymes. Reverse transcriptase-polymerase chain reactions (RT-PCR) with pollen-tube RNA and primers designed to conserved regions of bacterial and plant cellulose synthase (CesA) genes amplified a fragment that corresponded to an abundantly expressed cellulose-synthase-like gene named NaCslD1. A fragment from a true CesA gene (NaCesA1) was also amplified, but corresponding cDNAs could not be identified in a pollen-tube library, consistent with the very low level of expression of the NaCesA1 gene. RT-PCR with pollen-tube RNA and primers designed to regions conserved between the fungal FKS genes [that encode (1,3)-beta-glucan synthases] and their presumed plant homologs (the Gsl or glucan-synthase-like genes) amplified a fragment that corresponded to an abundantly expressed gene named NaGsl1. A second Gsl gene detected by RT-PCR (NaGsl2) was expressed at low levels in immature floral organs. The structure of full-length cDNAs of NaCslD1, NaCesA1, and NaGsl1 are presented. Both NaCslD1 and NaGsl1 are predominantly expressed in the male gametophyte (developing and mature pollen and growing pollen tubes), and we propose that they encode the catalytic subunits of two beta-glucan synthases involved in pollen-tube wall synthesis. Different beta-glucans deposited in one cell type may therefore be synthesized by enzymes from different gene families.

A Monogenic Recessive Gene, fw, Conditions Flower Doubling in Nicotiana alata

A double-flower form of Nicotiana alata Link & Otto was characterized genetically as a monogenic recessive trait expressed when homozygous. Reciprocal crosses demonstrated no maternal effect on expression of double flowers. A single dominant gene expressed in the homozygous or heterozygous state caused the single-flower phenotype. The symbol fw is proposed to describe the gene controlling double-flower phenotype.

Role of a callose synthase zymogen in regulating wall deposition in pollen tubes of Nicotiana alata Link et Otto

The callose synthase (CalS) activity of membrane preparations from cultured Nicotiana alata Link & Otto pollen tubes is increased several-fold by treatment with trypsin in the presence of digitonin, possibly due to activation of an inactive (zymogen) form of the enzyme. Active and inactive forms of CalS are also present in stylar-grown tubes. Callose deposition was first detected immediately after germination of pollen grains in liquid medium, at the rim of the germination aperture. During tube growth the 3-linked glucan backbone of callose was deposited at an increasing rate, reaching a maximum of 65 mg h−1 in tubes grown from 1 g pollen. Callose synthase activity was first detected immediately after germination, and then also increased substantially during tube growth. Trypsin caused activation of CalS throughout a 30-h time course of tube growth, but the degree of activation was higher for younger pollen tubes. Over a 10-fold range of callose deposition rates, the assayed CalS activity was sufficient to account for the rate of callose deposition without trypsin activation, implying that the form of CalS active in isolated membranes is responsible for callose deposition in intact pollen tubes. Sucrose-density-gradient centrifugation separated a lighter, intracellular membrane fraction containing only inactive CalS from a heavier, plasma-membrane fraction containing both active and inactive CalS, with younger pollen tubes containing relatively more of the inactive intracellular enzyme. The increasing rate of callose deposition during pollen-tube growth may thus be caused by the transport of inactive forms of CalS from intracellular membranes to the plasma membrane, followed by the regulated activation of these inactive forms in this final location.

Anatomical Analysis of Double-flower Morphogenesis in a Nicotiana alata Mutant

Anatomical analysis was performed using a double-flowered mutant of Nicotiana alata Link & Otto. Flower doubleness resulted from petaloid modification of the androecium. Vascularized petal-like outgrowths arose from the anther, connective, and filament of the stamen. The vasculature in petaloid outgrowths from the anther and upper part of the filament originated from and was continuous with the vascular bundle of the filament. In contrast, the vascular bundles formed in the outgrowths from the lower part of the filament developed independently of the vascular bundle of the filament and were not connected to it at any time. Emergences consisting of epidermal and ground parenchyma tissue and lacking vascularization arose from the filament.

Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto.

The callose synthase (UDP-glucose: 1,3-beta-D-glucan 3-beta-D-glucosyl transferase; EC 2.4.1.34) enzyme (CalS) from pollen tubes of Nicotiana alata Link et Otto is responsible for developmentally regulated deposition of the cell wall polysaccharide callose. Membrane preparations from N. alata pollen tubes grown in liquid culture were fractionated by density-gradient centrifugation. The CalS activity sedimented to the denser regions of the gradient, approximately 1.18 g.ml-1, away from markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and in membranes staining with phosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma membrane. Callose synthase activity from membranes enriched by downward centrifugation was solubilised with digitonin, which gave a 3- to 4-fold increase in enzyme activity, and the solubilised activity was then enriched a further 10-fold by product entrapment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that several polypeptides co-fractionated with CalS activity through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets.

Transformation of Nicotiana alata Link and Otto. with an Autoregulatory Senescence-inhibitor Gene Construct

Leaf explants of Nicotiana alata Link and Otto. were surface disinfested and cultured on Murashige and Skoog (MS) medium containing 2.66 μm N6-benzyladenine (BA) to promote shoot proliferation. After 5 weeks, proliferated shoots were removed and remaining callus saved. Callus was inoculated with Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase which catalyzes cytokinin synthesis. Following inoculation, the callus was cocultivated for 6 days on BA medium. Selection for transgenics was done on BA medium plus 100 mg Kanamycin and 400 mg Ticarcillin (antibiotics) per liter. Proliferating shoots were rooted on MS medium containing antibiotics. Rooted cuttings were transplanted to soil, acclimated and flowered in the greenhouse. Transgenics were outcrossed to a commercial N. alata hybrid. Seed was germinated in vitro on half-strength MS medium plus antibiotics. Segregation of transgenics to nontransgenics was 1:1. Evaluation of leaf senescence on 5-month-old plants showed 2 to 14 times fewer senesced leaves on the transgenic than the nontransgenic plants.

Localisation and expression of arabinogalactan-proteins in the ovaries of Nicotiana alata Link and Otto

The transmitting-tissue cells of the style of flowering plants secrete a complex extracellular matrix through which pollen tubes grow to the ovary to effect fertilisation. This matrix is particularly rich in a class of proteoglycans, the arabinogalactan-proteins (AGPs). AGPs from the ovary of Nicotiana alata were found to be developmentally regulated, as the different charge classes of AGPs altered during floral development. The AGPs from the mature ovary had charge characteristics that were distinct from those previously reported for the stigma and style. However, the concentration of AGP (0.6 μg/ml fresh weight) in the ovary did not change during development, or in response to either compatible or incompatible pollination. The AGPs of the ovary are mainly associated with the epidermis of the placenta.

The S-locus of Nicotiana alata: genomic organization and sequence analysis of two S-RNase alleles.

Genomic clones encoding the S2- and S6-RNases of Nicotiana alata Link and Otto, which are the allelic stylar products of the self-incompatibility (S) locus, were isolated and sequenced. Analysis of genomic DNA by pulsed-field gel electrophoresis and Southern blotting indicates the presence of only a single S-RNase gene in the N. alata genome. The sequences of the open-reading frames in the genomic and corresponding cDNA clones were identical. The organization of the genes was similar to that of other S-RNase genes from solanaceous plants. No sequence similarity was found between the DNA flanking the S2- and S6-RNase genes, despite extensive similarities between the coding regions. The DNA flanking the S6-RNase gene contained sequences that were moderately abundant in the genome. These repeat sequences are also present in other members of the Nicotianae.

Cloning and nucleotide sequence of the S7-RNase from Nicotiana alata Link and Otto.

Cloning and nucleotide sequence of the S7-RNase from Nicotiana alata Link and Otto.

Histochemistry and composition of the cell walls of styles of Nicotiana alata Link et Otto

The cell walls of styles of Nicotiana alata Link et Otto (ornamental tobacco; Solanaceae) were analysed chemically and examined histochemically. Cell-wall preparations were obtained from whole styles and from isolated transmitting-tissue cells. The style epidermal cells were shown histochemically to have thick, lignified secondary walls. These walls probably constituted a large proportion of the cell-wall preparation from whole styles as analysis of whole-style walls indicated that the major polysaccharides were xylans and cellulose, which are typical of lignified secondary walls of Magnoliopsida (dicotyledons). Lignification of the style epidermal walls was also demonstrated histochemically in 10 other species (5 genera including Nicotiana) of the sub-family Cestroideae of the Solanaceae, but not in 15 species (9 genera) of the sub-family Solanoideae of the Solanaceae, nor in 3 other species of dicotyledons and 2 species of Liliopsida (monocotyledons). Analysis of the cell-wall preparation from isolated transmitting-tissue cells of N. alata indicated that these contained cellulose, xyloglucans, and pectic polysaccharides, which is typical of primary cell walls of dicotyledons. However, the analysis indicated that the walls also contained an unusually high proportion of Type II arabinogalactans. Staining of the transmitting-tissue cell-wall preparation with β-glucosyl Yariv reagent, a histochemical reagent specific for arabinogalactan proteins, confirmed their presence, which may be related to the role of these cells in secreting the stylar extracellular matrix.

Uridine Diphosphate Glucose Metabolism and Callose Synthesis in Cultured Pollen Tubes of Nicotiana alata Link et Otto.

Membrane preparations from cultured pollen tubes of Nicotiana alata Link et Otto contain a Ca2+ -independent (1-3)-[beta]-D-glucan (callose) synthase activity that has a low affinity for UDP-glucose, even when activated by treatment with trypsin (H. Schlupmann, A. Basic, S.M. Read [1993] Planta 191: 470-481). Therefore, we investigated whether UDP-glucose was a likely substrate for callose synthesis in actively growing pollen tubes. Deposition of (1-3)-[beta]-glucan occurred at a constant rate, 1.4 to 1.7 nmol glucose min-1, in tubes from 1 mg of pollen from 3 h after germination; however, the rate of incorporation of radioactivity from exogenous [14C]-sucrose into wall polymers was not constant, but increased until at least 8 h after germination, probably due to decreasing use of internal reserves. UDP-glucose was a prominent ultraviolet-absorbing metabolite in pollen-tube extracts, with 1.6 nmol present in tubes from 1 mg of pollen, giving a calculated cytoplasmic concentration of approximately 3.5 mM. Radioactivity from [14C]-sucrose was rapidly incorporated into sugar monophosphates and UDP-glucose by the growing tubes, consistent with a turnover time for UDP-glucose of less than 1 min; the specific radioactivity of extracted UDP-[14C]glucose was equal to that calculated from the rate of incorporation of [14C]sucrose into wall glucans. Large amounts of less metabolically active neutral sugars were also present. The rate of synthesis of (1-3)-[beta]-glucan by nontrypsin-treated pollen-tube membrane preparations incubated with 3.5 mM UDP-glucose and a [beta]-glucoside activator was slightly greater than the rate of deposition of (1-3)-[beta]-glucan by intact pollen tubes. These data are used to assess the physiological significance of proteolytic activation of pollen-tube callose synthase.

A novel callose synthase from pollen tubes of Nicotiana

Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)-β-linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3-β-d-glucan 3-β-d-glucosyl transferase, EC 2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca2+ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (Km 1.5–2.5 mM); it was activated by β-glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca2+, and in the presence of 2 mM Ca2+ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (Km 0.45 mM); it also required a β-glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)-β-d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.

Influence of alkaloid precursors on the alkaloid content of Nicotiana alata root cultures

Root cultures of Nicotiana alata Link and Otto, growing in a Murashige and Skoog medium, contained the following alkaloid composition: nicotine (5 %), nornicotine (58%), anabasine (10%), and anatabine (27%), all with the (S)-configuration with high enantiomeric excess. When the root growth was carried out in the presence of increasing concentrations of [2-3H]nicotinic acid (0.002–5 mM), there were profound changes in the amounts and composition of the alkaloids after three days incubation. High concentrations of nicotinic acid inhibited both alkaloid formation and the demethylation of nicotine to nornicotine. Anatabine production was stimulated with increasing nicotinic acid concentration. A second series of experiments with a fixed concentration of [2-3H]nicotinic acid and increasing amounts of 1-methyl-Δ1-pyrrolinium chloride indicated that this latter compound stimulated nicotine production and also inhibited its demethylation. In a third set of experiments it was found that increasing concentrations of [2-14C]-Δ1-piperideine promoted the production of [2′-14C]anabasine and also stimulated nornicotine production.