Intact Tobacco Research Articles

Tobacco leaf tissue rapidly detoxifies direct salt loads without activation of calcium and SOS signaling.

Salt stress is a major abiotic stress, responsible for declining agricultural productivity. Roots are regarded as hubs for salt detoxification, however, leaf salt concentrations may exceed those of roots. How mature leaves manage acute sodium chloride (NaCl) stress is mostly unknown. To analyze the mechanisms for NaCl redistribution in leaves, salt was infiltrated into intact tobacco leaves. It initiated pronounced osmotically-driven leaf movements. Leaf downward movement caused by hydro-passive turgor loss reached a maximum within 2 h. Salt-driven cellular water release was accompanied by a transient change in membrane depolarization but not an increase in cytosolic calcium ion (Ca2+ ) level. Nonetheless, only half an hour later, the leaves had completely regained turgor. This recovery phase was characterized by an increase in mesophyll cell plasma membrane hydrogen ion (H+ ) pumping, a salt uptake-dependent cytosolic alkalization, and a return of the apoplast osmolality to pre-stress levels. Although, transcript numbers of abscisic acid- and Salt Overly Sensitive pathway elements remained unchanged, salt adaptation depended on the vacuolar H+ /Na+ -exchanger NHX1. Altogether, tobacco leaves can detoxify sodium ions (Na+ ) rapidly even under massive salt loads, based on pre-established posttranslational settings and NHX1 cation/H+ antiport activity. Unlike roots, signaling and processing of salt stress in tobacco leaves does not depend on Ca2+ signaling.

Qualitative Discrimination of Intact Tobacco Leaves Based on Near-Infrared Technology

To explore the application of near-infrared (NIR) technology to the quality analysis of raw intact tobacco leaves, a nondestructive discrimination method based on NIR spectroscopy is proposed. A “multiregion + multipoint” NIR spectrum acquisition method is developed, allowing 18 NIR diffuse reflectance spectra to be collected from an intact tobacco leaf. The spectral characteristics and spectral preprocessing methods of intact tobacco leaves are analyzed, and then different spectra (independent or average spectra) and different algorithms (discriminant partial least-squares (DPLS) and Fisher’s discriminant algorithms) are used to construct discriminant models for verifying the feasibility of intact leaf modeling and determining the optimal model conditions. Qualitative discrimination models based on the position, green-variegated (GV), and the grade of intact tobacco leaves are then constructed using the NIR spectra. In the application and verification stage, a multiclassification voting mechanism is used to fuse the results of multiple spectra from a single tobacco leaf to obtain the final discrimination result for that leaf. The results show that the position-GV discrimination model constructed using independent spectra and the DPLS algorithm and the grade discrimination model constructed using independent spectra and Fisher’s algorithm achieve optimal results with intact leaf NIR wavenumbers from 5006–8988 cm−1 and the first-derivative and standard normal variate transformation preprocessing method. Finally, when applied to new tobacco leaves, the position-GV model and the grade model achieve discrimination accuracies of 95.18% and 92.77%, respectively. This demonstrates that the two models have satisfactory qualitative discrimination ability for intact tobacco leaves. This study has established a feasible method for the nondestructive qualitative discrimination of the position, GV, and grade of intact tobacco leaves based on NIR technology.

Intercellular nuclear migration in cryofixed tobacco male meiocytes.

In this study, intercellular nuclear migration (INM), also known as cytomixis, was documented in cryofixed plant meiocytes for the first time. Intact tobacco inflorescences and flower buds as well as dissected individual anthers were cryofixed in liquid nitrogen by plunge freezing. Cryosubstituted and cryosectioned male meiocytes were analyzed by light microscopy. For cryosubstitution, the frozen material was kept in acetic alcohol at - 70°C for 1week. For cryosectioning, the frozen material was sectioned at - 20°C, and fixed with precooled acetic alcohol. Fixation of the intact tobacco inflorescences in Carnoy's solution was used as a control. Microscopy revealed good preservation of cell structure in the cryofixed anthers, flower buds, and inflorescences. INM was detectable in all the studied cryofixed and chemically fixed samples. The cytological picture of INM observed in the cryofixed meiocytes did not noticeably differ from the picture obtained with the chemically fixed cells. These results indicate that INM is observable irrespective of whether a physical or chemical fixation method is employed, with minimal damage from handling. Our results contradict the notion that INM is a phenomenon caused by mechanical, osmotic, or chemical artifacts during sample preparation.

Photoinjection of fluorescent nanoparticles into intact plant cells using femtosecond laser amplifier

The introduction of nanoparticles to intact plant cells is promising as a transporting technique of a wide range of functional molecules. Among various molecular delivery methods, femtosecond laser photoinjection possesses target selectivity at a single cell level and is potentially applicable for many types of materials. However, for plant cells, the vacuoles’ turgor pressure and the thick cell wall limit the application of photoinjection to only small objects. In this work, we overcome these limitations by employing a single pulse irradiation from a femtosecond laser amplifier. After laser irradiation on intact tobacco BY-2 cells, 80 nm fluorescent nanoparticles dispersed in a cell culture medium were successfully injected into their cytoplasm. This breakthrough would lead to a vast utilization of nanoparticles containing functional molecules for single cell manipulation in plant physiological study and genetic engineering. Such an injection was observed even when the laser pulse was focused neither on the cell wall nor on the cell membrane, but beside the cells. With these results, we suggest pore formation on the cell membrane by instantaneous deformation induced by an intense femtosecond laser pulse as an injection mechanism of nanoparticles. Reported photomechanical effects of the amplified femtosecond laser on the permeability of the biological membrane would offer new perspectives in biophotonics.

Implantable Organic Electronic Ion Pump Enables ABA Hormone Delivery for Control of Stomata in an Intact Tobacco Plant.

Electronic control of biological processes with bioelectronic devices holds promise for sophisticated regulation of physiology, for gaining fundamental understanding of biological systems, providing new therapeutic solutions, and digitally mediating adaptations of organisms to external factors. The organic electronic ion pump (OEIP) provides a unique means for electronically-controlled, flow-free delivery of ions, and biomolecules at cellular scale. Here, a miniaturized OEIP device based on glass capillary fibers (c-OEIP) is implanted in a biological organism. The capillary form factor at the sub-100 µm scale of the device enables it to be implanted in soft tissue, while its hyperbranched polyelectrolyte channel and addressing protocol allows efficient delivery of a large aromatic molecule. In the first example of an implantable bioelectronic device in plants, the c-OEIP readily penetrates the leaf of an intact tobacco plant with no significant wound response (evaluated up to 24 h) and effectively delivers the hormone abscisic acid (ABA) into the leaf apoplast. OEIP-mediated delivery of ABA, the phytohormone that regulates plant's tolerance to stress, induces closure of stomata, the microscopic pores in leaf's epidermis that play a vital role in photosynthesis and transpiration. Efficient and localized ABA delivery reveals previously unreported kinetics of ABA-induced signal propagation.

Agrobacterium-mediated Tnt1 mutagenesis of moss protonemal filaments and generation of stable mutants with impaired gametophyte.

The gametophyte of moss exhibits a simple body plan, yet its growth is regulated by complex developmental phenomena similar to angiosperms. Because moss can be easily maintained under laboratory conditions, amenable for gene targeting and the availability of genome sequence, P. patens has become an attractive model system for studying evolutionary traits. Until date, there has been no Agrobacterium-mediated Tnt1 mutagenesis protocol for haploid protonemal filaments of moss. Hence, we attempted to use the intact tobacco Tnt1 retrotransposon as a mutagen for P. patens. Bioinformatic analysis of initiator methionyl-tRNA (Met-tRNAi), a critical host factor for Tnt1 transposition process, suggested that it can be explored as a mutagen for bryophytes. Using protonemal filaments and Agrobacterium-mediated transformation, 75 Tnt1 mutants have been generated and cryopreserved. SSAP analysis and TAIL-PCR revealed that Tnt1 is functional in P. patens and has a high-preference for gene and GC-rich regions. In addition, LTR::GUS lines exhibited a basal but tissue-specific inducible expression pattern. Forward genetic screen resulted in 5 novel phenotypes related to hormonal and gravity response, phyllid, and gamete development. SSAP analysis suggests that the Tnt1 insertion pattern is stable under normal growth conditions and the high-frequency phenotypic deviations are possibly due to the combination of haploid explant (protonema) and the choice of mutagen (Tnt1). We demonstrate that Agrobacterium-mediated Tnt1 insertional mutagenesis could generate stable P. patens mutant populations for future forward genetic studies.

Gene Delivery to Tobacco Root Cells with Single-Walled Carbon Nanotubes and Cell-Penetrating Fusogenic Peptides.

Development of efficient, easy, and safe gene delivery methods is of great interest in the field of plant biotechnology. Considering the limitations of the usual transfection methods (such as transgene size and plant type), several new techniques have been tested for replacement. The success of some biological and synthetic nanostructures such as cell-penetrating peptides and carbon nanotubes in transferring macromolecules (proteins and nucleic acids) into mammalian cells provoked us to assess the ability of an engineered chimeric peptide and also arginine functionalized single-walled carbon nanotube in gene delivery to intact tobacco (Nicotiana tabacum var. Virginia) root cells. It was suggested that the engineered peptide with its special cationic and hydrophobic domains and the arginine functionalized single-walled carbon nanotube due to its nano-cylindrical shape can pass plant cell barriers while plasmid DNA (which codes green fluorescent protein) has been condensed on them. The success of gene delivery to tobacco root cells was confirmed by fluorescence microscopy and western blotting analysis.

Rapid and Simple Method for the Extraction of Genomic DNA from Tobacco (Nicotiana tabacum L.) Seedlings

Abstract Tobacco (Nicotiana tabacum L.) plants are known to present high levels of secondary metabolites that increase with the plant age. Molecular biology techniques like restriction enzyme digestion and PCR, requires as pre-requisite the isolation of genomic DNA of suitable purity, good quality and with low levels of contaminants. Several methods to isolate pure and intact tobacco DNA for molecular research purposes have been developed. In this work, a combination between a tobacco seed germination technique using gibberellic acid and a fast and simple genomic DNA extraction method from 14-days old tobacco seedlings to reduce the secondary metabolites levels in the final samples was presented. Ten tobacco genotypes were used to evaluate this method. The DNA concentrations were in a range between 0.73 μg/μL to 1.47 μg/μL for Habana-2000 cv. and Criollo cv., respectively. The absorbance ratios values to determine DNA quality were acceptable. This method allows the obtaining of high molecular weight DNA suitable for digestion with restriction enzymes, EcoRI and BamHI. Tobacco seedlings DNA in a short period of time, in a simple way and with a low cost, was obtained with this extraction method.

A Noninvasive Platform for Imaging and Quantifying Oil Storage in Submillimeter Tobacco Seed

While often thought of as a smoking drug, tobacco (Nicotiana spp.) is now considered as a plant of choice for molecular farming and biofuel production. Here, we describe a noninvasive means of deriving both the distribution of lipid and the microtopology of the submillimeter tobacco seed, founded on nuclear magnetic resonance (NMR) technology. Our platform enables counting of seeds inside the intact tobacco capsule to measure seed sizes, to model the seed interior in three dimensions, to quantify the lipid content, and to visualize lipid gradients. Hundreds of seeds can be simultaneously imaged at an isotropic resolution of 25 µm, sufficient to assess each individual seed. The relative contributions of the embryo and the endosperm to both seed size and total lipid content could be assessed. The extension of the platform to a range of wild and cultivated Nicotiana species demonstrated certain evolutionary trends in both seed topology and pattern of lipid storage. The NMR analysis of transgenic tobacco plants with seed-specific ectopic expression of the plastidial phosphoenolpyruvate/phosphate translocator, displayed a trade off between seed size and oil concentration. The NMR-based assay of seed lipid content and topology has a number of potential applications, in particular providing a means to test and optimize transgenic strategies aimed at the manipulation of seed size, seed number, and lipid content in tobacco and other species with submillimeter seeds.

FNOCT as a fluorescent probe for in vivo localization of nitric oxide distribution in tobacco roots

The nitric oxide-specific fluorescent probe Fluorescent Nitric Oxide Cheletropic Trap (FNOCT) 8a was applied to intact tobacco (Nicotiana tabacum cv. Samsun) roots to detect sites of nitric oxide formation and NO distribution. Three week old tobacco seedlings were gently removed from the sand culture pots with intact roots and transferred to small Petri dishes, whose base was replaced by a thin coverslip. Intact roots were subjected to FNOCT 8a to localize NO-dependent fluorescence in these roots; controls with an exogenous NO donor confirmed the presence and distribution of the probe in the roots. To confirm the NO-dependent fluorescence, roots were incubated with the three different NO scavengers cPTIO {2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-L-oxyl-3-oxide}, methylene blue and sodium diethyl dithiocarbamate (DCC) followed by incubation with FNOCT 8a. Methylene blue and DCC were able to completely quench NO-dependent fluorescence, cPTIO quenched partially. The roots were incubated in the presence of NaNO₂ and NaNO₃, which are substrates for nitrite:nitric oxide reductase (NI-NOR) and plasma membrane-bound nitrate reductase (PM-NR), respectively. The NO-dependent fluorescence was more or less same at the root tips upon treatment with NaNO₂, while the overall fluorescence was reduced in the presence of NaNO. Fluorescence from the living roots was visualized by inverted confocal laser scanning microscope (CLSM) using UV laser (excitation 360 nm and emission 408 nm). A specialized apparatus has been devised by the authors for analysis of intact roots as described in the methods section of this paper. Intact roots were chosen for microscopic observation rather than incised roots to avoid production of NO due to stress or physical injury.

Fungal elicitor-induced retardation and its restoration of root growth in tobacco seedlings

In this study, we investigated responses of growing and intact tobacco (N. tabacum cv Xanthi) seedlings to a fungal elicitor, a xylanase from Trichoderma viride (TvX). In addition to the induction of defense gene expression, TvX treatment caused the retardation of growth of seedlings. In the TvX-treated seedlings, growth of primary roots was markedly reduced through repression of cell division and longitudinal cell elongation in a meristematic zone and an elongation zone, respectively. However, cell differentiation to form vascular bundles and root hairs continued. In the TvX-treated root cap, disappearance of starch granules in columella cells and aggregation of border cells were observed. Furthermore, the TvX-induced growth retardation was restored after removal of the elicitor, resulting in a plastic alteration of root architecture. Therefore, the fungal elicitor might act as an environmental cue that regulates root growth and development as well as the ordinary defense responses in plant seedlings. These findings suggest a novel aspect of plant growth regulation via a plant–microbe interaction in the rhizosphere.

The effects of moderately high temperature on zeaxanthin accumulation and decay

Moderately high temperature reduces photosynthetic capacities of leaves with large effects on thylakoid reactions of photosynthesis, including xanthophyll conversion in the lipid phase of the thylakoid membrane. In previous studies, we have found that leaf temperature of 40°C increased zeaxanthin accumulation in dark-adapted, intact tobacco leaves following a brief illumination, but did not change the amount of zeaxanthin in light-adatped leaves. To investigate heat effects on zeaxanthin accumulation and decay, zeaxanthin level was monitored optically in dark-adapted, intact tobacco and Arabidopsis thaliana leaves at either 23 or 40°C under 45-min illumination. Heated leaves had more zeaxanthin following 3-min light but had less or comparable amounts of zeaxanthin by the end of 45min of illumination. Zeaxanthin accumulated faster at light initiation and decayed faster upon darkening in leaves at 40°C than leaves at 23°C, indicating that heat increased the activities of both violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). In addition, our optical measurement demonstrated in vivo that weak light enhances zeaxanthin decay relative to darkness in intact leaves of tobacco and Arabidopsis, confirming previous observations in isolated spinach chloroplasts. However, the maximum rate of decay is similar for weak light and darkness, and we used the maximum rate of decay following darkness as a measure of the rate of ZE during steady-state light. A simulation indicated that high temperature should cause a large shift in the pH dependence of the amount of zeaxanthin in leaves because of differential effects on VDE and ZE. This allows for the reduction in ΔpH caused by heat to be offset by increased VDE activity relative to ZE.

The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco

PevD1, a novel protein elicitor from the pathogenic cotton verticillium wilt fungus, Verticillium dahliae, induced a hypersensitive response in tobacco plants. In this paper, the elicitor was purified and analyzed using de novo sequencing. The protein-encoding pevD1 gene consists of a 468-bp open reading frame that produces a polypeptide of 155 amino acids, with a theoretical molecular weight of 16.23 kDa. The sequence of elicitor protein PevD1 was matched to the genomic sequence (GenBank accession no. ABJE 01000445.1) of a putative protein from V. dahliae strain vdls.17, but a function had not yet been reported. The pevD1 gene was expressed in Escherichia coli, and the recombinant protein was characterized for its ability to confer systemic acquired resistance to tobacco mosaic virus (TMV). Recombinant PevD1-treated plants exhibited enhanced systemic resistance compared to control, including a significant reduction in the number and size of TMV lesions on tobacco leaves. The elicitor protein-induced hydrogen peroxide production, extracellular-medium alkalization, callose deposition, phenolics metabolism, and lignin synthesis in tobacco. Our results demonstrate that elicitor-PevD1 triggers defense responses in intact tobacco plants.

Pulling Genetic RNA out of Tobacco Mosaic Virus Using Single-Molecule Force Spectroscopy

RNA-coat protein interactions in intact tobacco mosaic virus have been investigated for the first time directly on the single-molecule level by pulling the genetic RNA step by step out of the helical groove formed by its protein coat. The effects of pulling speed and pH on RNA-protein interactions are presented. In addition, the rebinding behavior of the detached RNA with the protein coat is discussed. Our results demonstrate the possibility of studying nucleic acid-protein interactions in more complicated systems using AFM-based single-molecule force spectroscopy.

Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor‐mediated defence responses in tobacco

A new protein elicitor, PeaT1, was purified from the mycelium of Alternaria tenuissima by column chromatography. PeaT1 was identified as a heat‐stable and acidic protein. It induced systemic acquired resistance to tobacco mosaic virus (TMV) in tobacco plants but did not cause hypersensitive response. The elicitor‐encoding gene was cloned by rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA is 624 bp in length and the open reading frame encodes for a polypeptide of 207 amino acids with a nascent polypeptide‐associated complex domain. The peaT1 gene was cloned into the expression vector pET‐28a and transformed into Escherichia coli BL21 (DE3). The recombinant elicitor also triggered defence responses in intact tobacco plants. The availability of the pure protein offers the possibility to isolate the corresponding receptor and links it to the downstream signalling pathway.

Moderate heat stress reduces the pH component of the transthylakoid proton motive force in light‐adapted, intact tobacco leaves

We measured the DeltaPsi and DeltapH components of the transthylakoid proton motive force (pmf) in light-adapted, intact tobacco leaves in response to moderate heat. The DeltaPsi causes an electrochromic shift (ECS) in carotenoid absorbance spectra. The light-dark difference spectrum has a peak at 518 nm and the two components of the pmf were separated by following the ECS for 25 s after turning the light off. The ECS signal was deconvoluted by subtracting the effects of zeaxanthin formation (peak at 505 nm) and the qE-related absorbance changes (peak at 535 nm) from a signal measured at 520 nm. Heat reduced DeltapH while DeltaPsi slightly increased. Elevated temperature accelerated ECS decay kinetics likely reflecting heat-induced increases in proton conductance and ion movement. Energy-dependent quenching (qE) was reduced by heat. However, the reduction of qE was less than expected given the loss of DeltapH. Zeaxanthin did not increase with heat in light-adapted leaves but it was higher than would be predicted given the reduced DeltapH found at high temperature. The results indicate that moderate heat stress can have very large effects on thylakoid reactions.

First Report of Bacterial Fruit Blotch of Watermelon Caused by Acidovorax avenae subsp. citrulli in Hungary.

Typical bacterial fruit blotch (BFB) symptoms were observed on cvs. Crisby, Suzy, Top Gun, and Lady watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai.) fruits in southeastern Hungary (Medgyesegyháza) in July 2007 when the mean maximum daytime temperature was greater than 32°C. Approximately 20 to 30 ha of watermelons were observed to be affected by the disease. Source of the infection was not determined, however, grafted watermelon transplants that were grown in this area had been imported from Turkey where the pathogen is present (2). Disease symptoms started with irregularly shaped, water-soaked lesions on the surface of the fruits. The lesions enlarged and the epidermis became brown and cracked. BFB symptoms were not readily visible on mature foliage. Colonies of the BFB pathogen were creamy white on nutrient agar (Difco, Detroit, MI). Strains were gram negative, oxidase positive, and produced acid from glucose aerobically. A cell suspension (50 μl of ~1 × 107 CFU/ml) from a 24-h nutrient plate culture was infiltrated with a hypodermic syringe into the intercellular spaces of fully developed intact tobacco (Nicotiana tabacum L. cv. White Burley) leaves to determine the hypersensitive reaction (HR) (1). A typical HR developed 20 h after leaf infiltration. Bacterial pathogenicity was tested on surface-sterilized, mature fruits of different plant species by injecting cell suspensions into the fruit tissues as previously described (each fruit was injected in five places; the negative control (sterile water) as well). Fruits were incubated for 7 days at 25°C and then observed for symptom development. Necrosis was observed at each point of inoculation with the pathogen for watermelon and green pepper (Capsicum annuum L.). Necrosis was also observed for cucumber (Cucumis sativus L.), zucchini (Cucurbita pepo L. convar. giromontiina Greb.), squash (C. pepo L.), and patisson (C. pepo L. convar. patissoniana Greb.). Necrosis was not observed when the pathogen was inoculated onto fruit of melon (Cucumis melo L.), tomato (Lycopersicon esculentum Mill.), and eggplant (Solanum melongena L.). Additionally, symptoms were not observed at the points inoculated with sterile water (negative control) for any of the fruits tested. To identify the pathogen, PCR was used with Acidovorax avenae subsp. citrulli-specific primers WFB1/2 (4). The 16s rDNA region amplified with a general bacterial primer pair (63f forward and 1389r reverse) (3) was cloned into a pBSK+ vector (Stratagene, La Jolla, CA) and sequenced by M13 forward and reverse primers (GenBank Accession No. AM850114). On the basis of the symptoms, biochemical tests (API 20NE; Biomérieux, Marcy l'Etoile, France), fatty acid methyl ester analysis (74.5 to 83.6% similarity), and 16SrDNA sequence homology (100% sequence similarity with AAC00-1), the pathogen was identified as A. avenae subsp. citrulli. To our knowledge, this is the first report of BFB of watermelon in Hungary.

Shoot Apex Demand Determines Assimilate and Nutrients Partitioning and Nutrient‐uptake Rate in Tobacco Plants

Abstract Our previous experiment revealed that apex‐removed plants have larger root systems but a lower K+‐uptake rates than intact tobacco plants. Since the apex is not only a center of growth and metabolism, but also an important place of auxin synthesis and export, the aims of this study were to distinguish whether the apex demand or auxin synthesized in the apex regulates assimilate and nutrients partitioning within plant, and to explain the reason for the lower K+‐uptake rate of the apex‐removed plant. In comparison with the control plant, covering the shoot apex with a black transparent plastic bag reduced net increases in dry matter and nutrients; however, the distribution of the dry matter and nutrients between shoot and roots and nutrient‐uptake rates were not changed. Removal of the shoot apex shifted the dry mass and nutrients distributions to roots, and reduced the rate of nutrient uptake. Application of 1‐naphthylacetic acid (NAA) could partly replace the role of the removed apex, stimulated assimilate and nutrient deposition into the treated tissue, and enhanced the reduced plasma membrane ATPase activity of roots to the control level. However, treatment of the apex‐removed plants with NAA could not rescue the reduced nutrient uptake rate and the shifted assimilates and nutrients partitioning caused by excision of the apex. Higher nutrient uptake rate of the intact plants could not be explained by root growth parameters, such as total root surface area and number of root tips. The results from the present study indicate that strong apex demand determined assimilates and nutrients partitioning and nutrient‐uptake rate in tobacco (Nicotiana tabacum) plants.

The Jasmonate-Induced Expression of the Nicotiana tabacum Leaf Lectin

Previous experiments with tobacco (Nicotiana tabacum L. cv Samsun NN) plants revealed that jasmonic acid methyl ester (JAME) induces the expression of a cytoplasmic/nuclear lectin in leaf cells and provided the first evidence that jasmonates affect the expression of carbohydrate-binding proteins in plant cells. To corroborate the induced accumulation of relatively large amounts of a cytoplasmic/nuclear lectin, a detailed study was performed on the induction of the lectin in both intact tobacco plants and excised leaves. Experiments with different stress factors demonstrated that the lectin is exclusively induced by exogeneously applied jasmonic acid and JAME, and to a lesser extent by insect herbivory. The lectin concentration depends on leaf age and the position of the tissue in the leaf. JAME acts systemically in intact plants but very locally in excised leaves. Kinetic analyses indicated that the lectin is synthesized within 12 h exposure time to JAME, reaching a maximum after 60 h. After removal of JAME, the lectin progressively disappears from the leaf tissue. The JAME-induced accumulation of an abundant nuclear/cytoplasmic lectin is discussed in view of the possible role of this lectin in the plant.

Sphingolipid metabolites selectively elicit increases in nuclear calcium concentration in cell suspension cultures and in isolated nuclei of tobacco

Sphingolipids are known to interfere with calcium-based signalling pathways. Here we report that these compounds modulate nuclear calcium signalling in tobacco BY-2 cells. Nuclear protein kinase activity phosphorylated endogenous sphingoid long-chain bases (LCBs), suggesting that LCBs are actively metabolized in the nucleus of tobacco BY-2 cells. The Δ4-unsaturated LCB d- erythro-sphingosine and the saturated LCB d- ribo-phytosphingosine elicited increases in free calcium in the nucleus in a dose-dependent and structure-related manner. However, neither sphingosine-1-phosphate nor C2-ceramide was able to stimulate nuclear calcium changes. N-, N-Dimethyl- d- erythro-sphingosine, a structural analogue of d- erythro-sphingosine, was the most efficient LCB so far tested in eliciting nuclear calcium changes both in intact tobacco BY-2 cells and in isolated nuclei. TRP channel inhibitors prevent the effect of DMS, suggesting that LCBs may activate TRP-like channels located on the inner nuclear membrane Collectively, the obtained data show that nuclei respond to LCBs on their own independently of the cytosolic compartment.