Localization of myosin on sperm-cell-associated membranes of tobacco (Nicotiana tabacum L.)

Summary Actomyosin interactions are reportedly the principal mechanism for the transport of nonmotile sperm cells of flowering plants inside the pollen tube and inside the embryo sac. Myosin has been demonstrated on the generative cell (the predecessor of sperm cells), although it is unclear from previous studies whether myosin is located directly on the plasma membrane of the male germ cells or on the external plasma membrane of the pollen cell that surrounds them. Immunogold scanning electron microscopy was used to localize myosin on isolated tobacco sperm cells, with and without associated membranes. When present, the pollen tube plasma membrane surrounding the sperm cells was labeled by an antimyosin antibody, as were pollen tube cytoplasmic organelles. Negligible labeling was observed directly on the plasma membrane of the sperm cells.     

Quantitative differences in sperm cells and organelles of tobacco (Nicotiana tabacum L.) grown under differing environmental conditions

In flowers grown at warm temperatures in environmental chambers and at cooler temperatures in the greenhouse, eight parameters of the sperm-cell organization of Nicotiana tabacum were examined during sperm cell maturation using serial ultrathin sectioning, transmission electron microscopy and quantitative cytology. Despite employing the same seed source, and similar soil and nutrient conditions, the surface area and volume of the cell, the nucleus and the chondriome were larger in flowers grown in growth chambers under warmer controlled conditions, whereas the number of plastids appeared to be the same, or slightly higher, in flowers grown under cooler greenhouse conditions. These results suggest that environmental conditions may influence the quantity of cytoplasmic organelles, including mitochondria and plastids, thus potentially influencing the likelihood of male cytoplasmic inheritance.     

Restoring full pollen fertility in transgenic male-sterile tobacco (Nicotiana tabacum L.) by Cre-mediated site-specific recombination

Producing hybrid seed requires an efficient pollination control system to prevent unwanted self-pollination. For further breeding, it would be advantageous to restore pollen fertility in the hybrids. In this work we demonstrate the use of tapetum-specific expression of a stilbene synthase (sts) transgene to induce pollen sterility in tobacco as has been shown previously. The sts-coding region was flanked by loxP recognition sites for Cre-recombinase. From 10 T₀-plants obtained, five proved to be male-sterile. They had smaller flowers with shorter stamina, but the vegetative phenotype was just as in the wild-type. Crossing male-sterile sts-plants with tobacco lines expressing the cre recombinase transgene resulted in site-specific recombination in the hybrids. GUS activity caused by fusion of the tap1-promoter with a promoterless gusA coding region indicated recombination events already in early stages of flower bud development. In all plants which had contained single or double sts-copies before crossing, these were excised, and pollen fertility was fully restored. The phenotype of these restored plants was as in wild-type controls. Contrary, from male sterile plants containing multiple copies of the sts-gene, not all copies were removed, and pollen sterility was maintained.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

Nonradioactive in situ localization of poly(A)+ RNA during pollen development in anthers of tobacco (Nicotiana tabacum L.)

Summary A method is described for non-radioactive labeling of total mRNA [poly(A)+ RNA] in plastic-embedded plant tissue sections. Oligo-deoxythymidylic acid (oligo-dT) labeled with digoxigenin-conjugated dUTP was used for in situ hybridization to poly(A)+ RNA in sections of tobacco (Nicotiana tabacum) anthers. The digoxigenin was immuno-stained using antidigoxigenin IgG and gold-labeled protein-A, followed by silver enhancement of the gold label. Reproducibly similar positive staining patterns were obtained with digoxigenin-labeled oligo-dT and polyuridylic acid [poly(U)], but not with a similarly labeled sense probe, poly(A). In the developing anthers, from the onset of meiosis to the production of pollen grains, labeling patterns were compatible with a gradual depletion of nuclear and chromosome-associated sporophytic mRNA molecules during prophase of meiosis, followed by postmeiotic production of gametophytic mRNA in microspore nuclei and the vegetative nuclei of the pollen grains.Abbreviations BSA bovine serum albumin - DIG digoxigenin - IgG immunoglobulin-G - oligo-dT oligo-deoxythymidylic acid - PAS-ABB periodic acid Schiff-aniline blue black - PBS phosphate buffered saline - poly(A) polyadenylic acid - poly(U) polyuridylic acid - SSC standard saline citrate     

Immobilization of cells of Nicotiana tabacum L. on polyphenyleneoxide coated with poly-L-lysine

Cells of Nicotiana tabacum L. cv. Wisconsin 38 were immobilized on poly (2,6-dimethyl)-p-phenyleneoxide in powder form (Sorfix) coated with poly-L-lysine (molecular weight 40 000 daltons). The dependence of cell immobilization on the amount of bound polyL-lysine was estimated.Abbreviations MW molecular weight - dwt dry weight - fwt fresh weight     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.     

Zearalenone and flower bud formation in thin-cell layers of Nicotiana tabacum L.

Three lines of evidence indicated a connectionbetween zearalenone (ZEN) and flower bud formationin thin cell layer (TCL) explants of Nicotianatabacum L. cv. Samsun. (1) There were two peaks inthe endogenous ZEN level during the formation offlower buds. (2) The specific inhibitor of ZENbiosynthesis, malathion (MAL), inhibited thebiosynthesis of endogenous ZEN and at the same timeflower bud neoformation. (3) Exogenous ZEN inducedflower bud neoformation.     

Studies on the relationship between ploidy level,morphology, the concentration of some phytohormones and the nicotine concentration of haploid and doubled haploid tobacco (Nicotiana tabacum L.) and NICA plants

As compared to doubled haploid plants of the same origin, haploid tobacco plants are characterized by narrow leaves and in these leaves the endogenous concentration of gibberellins was considerably higher than in doubled haploids. This higher GA activity is almost entirely due to elevated levels of polar gibberellins. The same leaf shape as in haploids could be induced by GA₃ sprays to doubled haploids. A similar leaf shape was also observed on tissue culture derived so called NICA plants displaying the morphology of tobacco plants as described by Dudits et al. (1987) from whom the plant material was obtained as a gift. Here, in the leaves of a special strain with narrow lamina again a much higher gibberellin activity was detected than in the leaves of plants of the original tobacco strain. Histochemical determination of the relative DNA content indicated that leaves of NICA were chimaeras containing 1C cells besides cells with higher C values. Obviously, haploidy is somehow related to the endogenous gibberellin activity in tobacco plant material with consequences on the morphological appearance of 1n plants. Comparing some haploid and doubled haploid strains in tissue culture and pot and field experiments in several years apparently the genotype of the plant material is more significant for nicotine concentration than the ploidy level.Abbreviations DW dry weight - FW fresh weight - LSI leaf shape index     

In vitro induction of stigma-like and style-like structures from receptacle tissue in Nicotiana tabacum L.

Stigma-like and style-like structures were induced from the receptacle tissue in tobacco (Nicotiana tabacum L.). The presence of kinetin was necessary to induce these structures. The structures have a form similar to and the same function as normal stigmas and styles.     

Characterisation and immunolocation of an 87 kDa polypeptide associated with UDP-glucuronic acid decarboxylase activity from differentiating tobacco cells (Nicotiana tabacum L.)

UDP-glucuronic acid decarboxylase catalyses the reaction responsible for the formation of UDP-xylose and commits assimilate for the biosynthesis of cell wall polysaccharides and glycosylation of proteins. Xylose-rich polymers such as xylans are a feature of dicot secondary walls. Thus a cell culture system of tobacco transformed with the ipt gene from Agrobacterium tumefaciens for cytokinin production and which when manipulated with auxin and sucrose leads to induction of xylogenesis, has been used as a source for purification of the enzyme. UDP-glucuronic acid decarboxylase was purified by ion-exchange, gel filtration and affinity chromatography on Reactive Brown-Agarose. The native enzyme had an apparent M(r) of 220,000 which yielded a single subunit of 87,000 when analysed on SDS-PAGE using silver staining. This appears to be a novel form of the enzyme since a gene family encoding polypeptides around M(r) 40,000 with homology to the fungal enzyme also exists in plants. Using an antibody raised to the native 87 kDa form of the enzyme, this decarboxylase was localised mainly to to cambium and differentiating vascular tissue in tobacco stem, consistent with a role in the provision of UDP-xylose for the synthesis of secondary wall xylan. Further analysis using immunogold electron microscopy localised the 87 kDa UDP-glucuronic acid decarboxylase to the cytosol of developing vascular tissue.     

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.     

Further characterization of picloram-tolerant mutants of Nicotiana tabacum

Summary A genetic and preliminary biochemical analysis has been performed on four picloram-tolerant mutants of Nicotiana tabacum that were isolated from cell cultures. The four mutations define three distinct linkage groups. Mutant seedlings incorporate radioactively labeled picloram normally and do not modify or degrade the herbicide in a manner that alters its solubility characteristics.     

Somatic embryogenesis in Nicotiana tabacum L.: induction by thidiazuron of direct embryo differentiation from cultured leaf discs

Summary Induction of somatic embryogenesis by different growth regulators was examined in leaf disc cultures of Nicotiana tabacum L. Direct differentiation of somatic embryos occurred on media supplemented with naphthaleneacetic acid (NAA) and N⁶-benzylaminopurine (BAP). Thidiazuron (N-phenyl-N-1, 2,3,-thiadiazol-5-ylurea; TDZ) not only substituted for the most effective NAA-BAP combination but also induced a higher frequency of somatic embryogenesis. Regenerated somatic embryos were capable of developing into plants.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - NAA Naphthaleneacetic acid - TDZ N-phenyl-N-1,2,3,- thiadiazol-5-ylurea     

Carbon metabolism enzymes and photosynthesis in transgenic tobacco (Nicotiana tabacum L.) having excess phytochrome

J.M. Keller et al. (1989, EMBO J. 8, 1005–1012) introduced a phytochrome gene controlled by a cauliflower mosaic virus 35S promoter into tobacco (Nicotiana tabacum L.) providing material to test whether several photosynthesis enzymes can be increased by one modification to the plant. We report here that this transgenic tobacco had greater amounts of all enzymes examined as well as greater amounts of total protein and chlorophyll per unit leaf area. Fructose bisphosphatase (E.C. 3.1.3.11), glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.12), and sucrose-phosphate synthase (E.C. 2.4.1.14) were also higher when expressed per unit protein. However, ribulose-1,5-bisphosphate carboxylase (E.C. 4.1.1.39) amount per unit leaf protein was the same in transgenic and wild-type (WT) plants. Photosynthesis in the transgenic plants was lower than in WT at air levels of CO₂, but higher than in WT above 1000 bar CO₂. The photosynthesis results indicated a high resistance to CO₂ diffusion in the mesophyll of the transgenic plants. Examination of electron micrographs showed that chloroplasts in the transgenic plants were often cup-shaped, preventing close association between chloroplast and cell surface. Chloroplast cupping may have caused the increase in the mesophyll resistance to CO₂ diffusion. We conclude that it is possible to affect more than one enzyme with a single modification, but unexpected physical modifications worsened the photosynthetic performance of this plant.Abbreviations CABP 2-carboxyarabitinol 1,5-bisphosphate - FBP fructose-1,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - GAP glyceraldehyde 3-phosphate - Rubisco ribulose-1,5-bisphosphate carboxylase - SPS sucrose-phosphate synthase - WT wild type This research was supported by U.S. Department of Energy contracts DE-FG02-87ER60568 to T.D.S. and DE-FG02-88ER 13968 to R.D.V. We thank Drs. Joel Cherry and Howard P. Hershey for assistance with the transgenic plants.     

Is bromodeoxyuridine resistance a consequence of cytokinin habituation in Nicotiana tabacum?

Cytokinin-habituated, as compared to nonhabituated Nicotiana tabacum cv. Havana 425 callus was shown to tolerate higher concentrations of 5-bromodeoxyuridine (BUdR) if cytokinin was added to the medium [Meins, Planta 129, 239–244 (1976)].Our aim was to clarify whether or not BUdR-resistance and cytokinin-habituation are linked characters in callus cultures of our BUdR-resistant tobacco mutant BR 37/21, obtained from Nicotiana tabacum cv. Ottawa.It is demonstrated that BUdR-resistance and cytokinin-habituation are independent characters in the case of this mutant. Non-habituated BR 37/21 callus grows as a resistant line on a medium containing BUdR at a selective concentration (30 mg/l) whereas cytokinin-habituated callus, not selected for BUdR-resistance, behaves as a sensitive line, i.e. turns brown and dies, on the same medium.     

Altered feedback sensitivity of acetohydroxyacid synthase from valine-resistant mutants of tobacco (Nicotiana tabacum L.)

Acetohydroxyacid synthase (EC 4.1.3.18) has been extracted from leaves of three valine-resistant (Val^r) tobacco (Nicotiana tabacum) mutants, and compared with the enzyme from the wild-type. The enzyme from all three mutants is appreciably less sensitive to inhibition by leucine and valine than the wild-type. Two of the mutants, Val^r-1 and Val^r-6, have very similar enzymes, which under all conditions are inhibited by less than half that found for the wild-type. The other mutant, Val^r-7, has an enzyme that only displays appreciably different characteristics from the wild-type at high pyruvate or inhibitor concentrations. Enzyme from Val^r-7 also has a higher apparent K_m for pyruvate, threefold greater than the value determined for the wild-type and the other mutants. The sulphonylurea herbicides strongly inhibit the enzyme from all the lines, though the concentrations required for half-maximal inhibition of enzyme from Val^r-1 and Val^r-6 are higher than for Val^r-7 or the wildtype. No evidence has been found for multiple isoforms of acetohydroxyacid synthase, and it is suggested that the valine-resistance of these mutant lines is the result of two different mutations affecting a single enzyme, possibly involving different subunits.     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

Influence of p-dimethylaminoazobenzene and doxorubicin on tobacco cell growth (Nicotiana tabacum L.)

Callus growth, in tobacco pith tissue culture, is activated by p-dimethylaminoazobenzene (p-DAAB), whose carcinogenic properties are well known. Ultrastructural changes, appearing in a period prior to initiation of cell proliferation, occur earlier and more intensely in the presence of the carcinogen. This also influences changes in non-histone chromatin proteins (NHCP). Doxorubicin (DR), an anti-tumor drug, inhibits callus growth and modifies the pattern of NHCP.