Intact Tobacco Plants Research Articles

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.

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.

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.

Inhibition of Ethylene Biosynthesis Enhances Vegetative Bud Formation without Affecting Growth and Development of Transgenic Tobacco Plants

The role of ethylene in vegetative bud formation was investigated using transgenic tobacco plants expressing an antisense tomato 1-aminocyclopropane-carboxylic acid synthase (ACS) gene. Northern blot hybridization showed that the accumulation of ACS mRNA was strongly reduced in the bud-forming leaf explants of the transgenic plants. Consequently, these transgenic tissues exhibited low ACS enzyme activity, 1-aminocyclopropane-carboxylic acid (ACC) content and ethylene production, and at the same time the tissue capacity to generate buds was greatly enhanced. However, it was also noted that the antisense ACS gene did not inhibit the endogenous ACS gene expression in intact transgenic tobacco plants. The growth and development of the transgenic tobacco was almost identical to control plants with respect to height, internode number, leaf morphology, and flowering time. Furthermore, mature leaves of transgenic tobacco had similar chlorophyll content, stomatal conductance, photosynthetic ability, and transpiration rates compared to control plants. These results demonstrated that ethylene plays an important role in bud formation in tobacco tissue culture.

Homology-dependent DNA transfer from plants to a soil bacterium under laboratory conditions: implications in evolution and horizontal gene transfer.

DNA transfer was demonstrated from six species of donor plants to the soil bacterium, Acinetobacter spp. BD413, using neomycin phosphotransferase (nptII) as a marker for homologous recombination. These laboratory results are compatible with, but do not prove, DNA transfer in nature. In tobacco carrying a plastid insertion of nptII, transfer was detected with 0.1 g of disrupted leaves and in oilseed rape carrying a nuclear insertion with a similar quantity of roots. Transfer from disrupted leaves occurred in sterile soil and water, without the addition of nutrients. It was detected using intact tobacco leaves and intact tobacco and Arabidopsis plants in vitro. Transfer was dose-dependent and sensitive to DNase, and mutations in the plant nptII were recovered in receptor bacteria. DNA transfer using intact roots and plants in vitro was easily demonstrated, but with greater variability. Transfer varied with plant genome size and the number of repeats of the marker DNA in the donor plant. Transfer was not detected in the absence of a homologous nptII in the receptor bacteria. We discuss these results with reference to non-coding DNA in plant genomes (e.g., introns, transposons and junk DNA) and the possibility that DNA transfer could occur in nature.

Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes.

Tolerance against oxidative stress generated by high light intensities or the catalase inhibitor aminotriazole (AT) was induced in intact tobacco plants by spraying them with hydrogen peroxide (H2O2). Stress tolerance was concomitant with an enhanced antioxidant status as reflected by higher activity and/or protein levels of catalase, ascorbate peroxidase, guaiacol peroxidases, and glutathione peroxidase, as well as an increased glutathione pool. The induced stress tolerance was dependent on the dose of H2O2 applied. Moderate doses of H2O2 enhanced the antioxidant status and induced stress tolerance, while higher concentrations caused oxidative stress and symptoms resembling a hypersensitive response. In stress-tolerant plants, induction of catalase was 1.5-fold, that of ascorbate peroxidase and glutathione peroxidase was 2-fold, and that of guaiacol peroxidases was approximately 3-fold. Stress resistance was monitored by measuring levels of malondialdehyde, an indicator of lipid peroxidation. The levels of malondialdehyde in all H2O2-treated plants exposed to subsequent high light or AT stress were similar to those of unstressed plants, whereas lipid peroxidation in H2O2-untreated plants stressed with either high light or AT was 1.5- or 2-fold higher, respectively. Although all stress factors caused increases in the levels of reduced glutathione, its levels were much higher in all H2O2-pretreated plants. Moreover, significant accumulation of oxidized glutathione was observed only in plants that were not pretreated with H2O2. Extending the AT stress period from 1 to 7 days resulted in death of tobacco plants that were not pretreated with H2O2, while all H2O2-pretreated plants remained little affected by the prolonged treatment. Thus, activation of the plant antioxidant system by H2O2 plays an important role in the induced tolerance against oxidative stress.

Diurnal changes in nitrogen assimilation of tobacco roots.

To gain an insight into the diurnal changes of nitrogen assimilation in roots the in vitro activities of cytosolic and plasma membrane-bound nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.7.7.1) and cytosolic and plastidic glutamine synthetase (EC 6.3.1.2) were studied. Simultaneously, changes in the contents of total protein, nitrate, nitrite, and ammonium were followed. Roots of intact tobacco plants (Nicotiana tabacum cv. Samsun) were extracted every 3 h during a diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase were active throughout the day-night cycle. Two temporarily distinct peaks of nitrate reductase were detected: during the day a peak of soluble nitrate reductase in the cytosol, in the dark phase a peak of plasma membrane-bound nitrate reductase in the apoplast. The total activities of nitrate reduction were similar by day and night. High activities of nitrite reductase prevented the accumulation of toxic amounts of nitrite throughout the entire diurnal cycle. The resulting ammonium was assimilated by cytosolic glutamine synthetase whose two activity peaks, one in the light period and one in the dark, closely followed those of nitrate reductase. The contribution of plastidic glutamine synthetase was negligible. These results strongly indicate that nitrate assimilation in roots takes place at similar rates day and night and is thus differently regulated from that in leaves.

N-Acylethanolamines in signal transduction of elicitor perception. Attenuation Of alkalinization response and activation of defense gene expression

In a recent study of N-acylphosphatidylethanolamine (NAPE) metabolism in elicitor-treated tobacco (Nicotiana tabacum L.) cells, we identified a rapid release and accumulation of medium-chain N-acylethanolamines (NAEs) (e.g. N-myristoylethanolamine or NAE 14:0) and a compensatory decrease in cellular NAPE (K.D. Chapman, S. Tripathy, B. Venables, A.D. Desouza [1998] Plant Physiol 116: 1163-1168). In the present study, we extend this observation and report a 10- to 50-fold increase in NAE 14:0 content in leaves of tobacco (cv Xanthi) plants treated with xylanase or cryptogein elicitors. Exogenously supplied synthetic NAE species affected characteristic elicitor-induced and short- and long-term defense responses in cell suspensions of tobacco and long-term defense responses in leaves of intact tobacco plants. In general, synthetic NAEs inhibited elicitor-induced medium alkalinization by tobacco cells in a time- and concentration-dependent manner. Exogenous NAE 14:0 induced expression of phenylalanine ammonia lyase in a manner similar to fungal elicitors in both cell suspensions and leaves of tobacco. NAE 14:0, but not myristic acid, activated phenylalanine ammonia lyase expression at submicromolar concentrations, well within the range of NAE 14:0 levels measured in elicitor-treated plants. Collectively, these results suggest that NAPE metabolism, specifically, the accumulation of NAE 14:0, are part of a signal transduction pathway that modulates cellular defense responses following the perception of fungal elicitors.

A rapid and easy method of detecting levels of gene expression in intact tobacco plants using an Agrobacterium-mediated transient assay: Toward development of nutritional diagnosis based on gene expression

For the use of gene expression for nutritional diagnosis, a rapid and easy method of detecting levels of gene expression was proposed. Agrobacterium tumefaciens carrying the GUS reporter gene was injected into intact tobacco leaves with a plastic syringe, and transient GUS expression was determined. GUS activities were reproducible in young leaves.

Induction of somatic DNA damage as measured by single cell gel electrophoresis and point mutation in leaves of tobacco plants

The induction and measurement of DNA damage in nuclei of plant tissues is a new area of study with the alkaline single cell gel electrophoresis/comet assay. Methods to isolate plant cell nuclei cause high levels of DNA damage which are detected by the comet assay. We developed a method to isolate nuclei from leaf tissue of Nicotiana tabacum (a 1 +/a 1; a 2 +/a 2) in a modified Sörensen buffer that resulted in constant, low tail moment values for the negative controls. After treating intact tobacco plants with 1–8 mM ethyl methanesulfonate (EMS) we obtained a direct concentration-response with an average median tail moment of 65.9±4.4 μm for plants exposed to the highest EMS concentration as compared to the median control tail moment value of 4.1±0.8. We found that the highest resolution was obtained with electrophoretic conditions of 0.74 V/cm at 300 mA for 20 min. Multiple leaves could be analyzed per plant within each treatment group and the tail moments were not significantly different. Tobacco seedlings were treated with EMS in the same manner as used for the comet assay and mutations were induced in the leaf primordia. The mean mutant frequency for the control was 1.46±0.20 mutant sectors/leaf. The mutant frequency increased in a concentration dependent manner; the mutant frequency induced by 8 mM EMS was 37.89±2.37 mutant sectors/leaf. The comet tail moment values and the leaf mutant frequency were highly correlated ( r=0.98). The genetic response factor was calculated by the ratio of the difference in the response within the linear portion of each concentration-response curve divided by the slope of the curve. The genetic response factor for the tail moment was 7.82 while the value for mutation induction was 7.76. In this paper we describe a sensitive method with high resolution to apply the alkaline comet assay to plant leaves. The comet assay response was compared to that of induced point mutation. With this sensitive method for nuclei isolation from plant leaves, the alkaline SCGE assay could be incorporated into in situ plant environmental monitoring.

Distribution of hydroxycinnamoyl-CoA: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase in higher plants

The occurrence of hydroxycinnamoyl-CoA: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase in roots of plants from various families is reported. Among the 21 species investigated, 17 plants representing 11 different families of angiosperms and gymnosperms showed extractable activity. In intact tobacco plants, the enzyme which was undetectable in leaves and stems, was apparently restricted to roots. Relatively high enzymatic activity was found however in cell suspension cultures grown from tobacco pith.

Influence of glutathione (GSH) on net uptake of sulphate and sulphate transport in tobacco plants

Net uptake of sulphate by the roots, and its transport to the shoot was inhibited in intact tobacco plants by 1 h exposure of its roots to 0.1 or 1.0 mM GSH. The relative amount of sulphate taken up that was transported to the shoot was not affected by this treatment. Apparently, sulphate uptake, but not xylem loading of sulphate, was inhibited by GSH. Similar results were obtained when L-cysteine was used instead of GSH. As L-cysteine, mediated inhibition of sulphate, net uptake was counteracted by exposure of the roots to BSO, GSH synthesis seems to be required for inhibition of sulphate uptake by L-cysteine. However, exposure to 0.1 or 1.0mM GSH did not result In an enhanced GSH level in roots or shoots. Also the cysteine contents remained unchanged. Small changes in metabolically active pools of GSH or cysteine seem to be responsible for the inhibition of sulphate uptake and its transport to the shoot. Flap-feeding of GSH to a mature leaf resulted in an inhibition of net uptake of sulphate by the roots and its transport to the shoot. These findings support the hypothesis that GSH produced in the leaves acts as a signal to control sulphur nutrition of plants.

Regulation of the expression of rbcS and other photosynthetic genes by carbohydrates: a mechanism for the ‘sink regulation’ of photosynthesis?

SummaryThese experiments were carried out to investigate whether accumulation of carbohydrate leads to decreased expression of genes involved in photosynthesis. Addition of glucose to autotrophic cell suspension cultures of Chenopodium led to a large and reversible decrease of the steady state transcript levels of rbcS, cab and atp‐& within 5 h, but did not decrease 18S rRNA or transcript for two glycolytic enzymes. Run‐on transcription in isolated nuclei showed that transcription rate had been decreased. [35S]Methionine feeding showed that de novo synthesis of Rubisco was inhibited. Decreased rbcS transcript was also found after feeding glucose to detached leaves, and in transgenic plants expressing invertase in the apoplast to inhibit phloem transport, and in leaves on intact tobacco and potato plants which were cold‐girdled to decrease export. The decrease of rbcS transcript level occurred within 12 h of coldgirdling. Comparison of carbohydrate content and rbcS transcript level indicated that carbohydrate content per se is not the direct signal for regulation of gene expression. Feeding of transported analogues indicates that metabolism rather than transport of the sugars is required. Over‐expression of rbcS was found in low CO2, again indicating metabolic control of expression. It is proposed that photosynthetic gene expression is inhibited by metabolic factors related to high carbohydrate content, and that this represents a basic mechanism for the ‘sink regulation’ of photo‐synthesis.

Regulation of the expression of rbcS and other photosynthetic genes by carbohydrates: a mechanism for the 'sink regulation' of photosynthesis?

These experiments were carried out to investigate whether accumulation of carbohydrate leads to decreased expression of genes involved in photosynthesis. Addition of glucose to autotrophic cell suspension cultures of Chenopodium led to a large and reversible decrease of the steady state transcript levels of rbcS, cab and atp-& within 5 h, but did not decrease 18S rRNA or transcript for two glycolytic enzymes. Run-on transcription in isolated nuclei showed that transcription rate had been decreased. [35S]Methionine feeding showed that de novo synthesis of Rubisco was inhibited. Decreased rbcS transcript was also found after feeding glucose to detached leaves, and in transgenic plants expressing invertase in the apoplast to inhibit phloem transport, and in leaves on intact tobacco and potato plants which were cold-girdled to decrease export. The decrease of rbcS transcript level occurred within 12 h of coldgirdling. Comparison of carbohydrate content and rbcS transcript level indicated that carbohydrate content per se is not the direct signal for regulation of gene expression. Feeding of transported analogues indicates that metabolism rather than transport of the sugars is required. Over-expression of rbcS was found in low CO2, again indicating metabolic control of expression. It is proposed that photosynthetic gene expression is inhibited by metabolic factors related to high carbohydrate content, and that this represents a basic mechanism for the ‘sink regulation’ of photo-synthesis.

Investigations on the Inhibition of Potato Virus X by Some Amino Acid Analogues

Summary Ethionine markedly inhibits the replication of potato virus X (PVX) after infiltration into leaf disks of Nicotiana tabacum L. “Samsun”. P-fluorophenylalanine and 5-methyl-DL-tryptophane inhibit this replication less effectively and only at phytotoxic concentrations. Methionine strongly reduces the inhibitory activity of ethionine in the replication of PVX. In leaf disks with synchronized virus infection, the time-response course of ethionine inhibition indicates that a late event is inhibited. Short time after the beginning of ethionine inhibition decline virus protein was detected by ELISA. Contrary to leaf disks, in intact tobacco plants PVX replication is inhibited by ethionine sprays only in very high and phytotoxic doses. However, low doses of ethionine increase synergistically the inhibitory activity of 5-azadihydrouracil.

Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants.

A cauliflower mosaic virus (CaMV) 35S promoter derivative, which is tightly repressed by the Tn 10 encoded Tet repressor in a transient expression system as well as in transgenic plants has been constructed. After treatment of transgenic plants with tetracycline (Tc) the activity of the reporter enzyme beta-glucuronidase (GUS) increased up to 500-fold in tissue culture as well as under greenhouse conditions. Efficient de-repression was achieved by Tc uptake through the roots as well as by Tc treatment of leaves of intact plants. As Tc is not very stable in the plants, this system can also be used for a transient expression of a transgene. This system provides a unique tool for regenerating transgenic plants carrying a repressed transgene and for efficiently de-repressing its activity by a specific inducer at any time point of further development.

Measurement of Formaldehyde, Hydrogen Peroxide and Non-protein Thiols in Tobacco Leaves during Ageing

Summary Formaldehyde, hydrogen peroxide and non-protein thiols were measured in the leaves of intact tobacco plants at 3 ageing stages as the function of plant age and leaf position. The average formaldehyde level changed only slightly during ageing within the range 1.1–1.4 μmole · (g fresh weight)-1. The highest formaldehyde levels were found in the youngest leaves. The H2O2 level depended strongly on plant age and leaf position. The average H2O2 content of leaves was 3.3 μmole · (g fresh weight)-1 in the oldest and in the middle-aged plants, while this value was 2.3 in the youngest plants. Within one plant gradually increasing H2O2 levels were found from the apex toward the base, but it declined in the oldest, yellowing leaves. The maximum values of H2O2 level were 2.9, 4.4 and 5.2 μmole · (g fresh weight)-1 in the young, middle-aged and old age-groups, respectively. The peroxidase activity increased more significantly with the plant age than the H2O2 level. Reversely, the highest catalase activities were found in the young leaves. Gradually decreasing foliar thiol contents were found from the apex towards the base. The reverse changes of H2O2 and thiol concentration in the leaves refer to the alteration of cellular redox potential during ageing.

The effects of auxin and antiauxin in an in vitro bioassay of flower regulatory activity in leaf exudate from tobacco plants

An in vitro bioassay system for floral bud formation has been established using Nicotiana tabacum L. cv. MC, explants excised from floral stalks cultured on modified Murashige‐Skoog medium containing excess auxin and antiauxin. Three auxins. indolyl‐3‐acetic acid (IAA), 4‐chloroindolyl‐3‐acetic acid and 5,6‐dichloroindolyl‐3‐acetic acid, were tested for floral bud‐forming activity; IAA was most efficient. Three antiauxins. 5,7‐dichlorindolyl‐3‐isobutyric acid (5,7‐Cl2‐HBA), p‐chlorophenoxyiso butyric acid and 2,3,5‐triiodobenzoic acid, were tested for the ability to reverse the inhibition of floral bud formation caused by excess IAA. Only 5,7‐Cl2‐HBA was very effective. Leaf exudates from short day‐ and long day‐treated tobacco plants were added to the bioassay system which contained 1 µM 6‐benzylaminopurine, 5 µM IAA and 5 µM 5, 7‐Cl2‐HBA, Interestingly, only the leaf exudate from short day‐treated plants stimulated floral bud formation. Moreover, the buds produced grew into large. well‐developed ones with shapes similar to those of the natural floral buds of intact tobacco plants.

5'-dephosphorylated 2',5'-adenylate trimer and its analogs. Inhibition of tobacco mosaic virus replication in tobacco mosaic virus-infected leaf discs, protoplasts, and intact tobacco plants.

The effect of the 5'-dephosphorylated 2',5'-adenylate trimer and its 2',5'-trimer core analogs on the inhibition of tobacco mosaic virus (TMV) replication was determined in tobacco leaf discs, protoplasts, and whole tobacco plants, using infectivity tests and enzyme-linked immunosorbent assays. A structure-activity-metabolic stability-toxicity analysis of the 2',5'-adenylate trimer core molecule in TMV-infected Nicotiana glutinosa was determined. Modification at either the 6-amino position of the adenylate residues (i.e. inosinate trimer core) or at the 2' terminus (i.e. A-A-ara-A or A-A-Tu) inhibited replication of TMV. Modification of the 3'-hydroxyl group of the adenylate residues to 3-deoxyribose (i.e. the 2',5'-cordycepin trimer core) inhibited TMV replication better than the 2',5'-adenylate trimer core molecule. With enzyme-linked immunosorbent assays, there was complete inhibition of TMV replication by 200 nM 2',5'-adenylate trimer core for 60 h and by 200 nM 2',5'-cordycepin trimer core for 96 h. The amount of 2',5'-oligonucleotides associated with the leaves was determined using 2',5'-[3H]cordycepin trimer core; 1 X 10(-12) mol/cm2 of plant leaves inhibited TMV replication by 99%. No 2',5'-phosphodiesterase activity was detected in TMV-infected and noninfected leaf extracts. Therefore, the 2',5'-trimer cores were potent inhibitors of TMV replication at nanomolar concentrations, i.e. at 1000-fold lower concentration than that required in mammalian systems.