Inheritance of Altered Flower Morphology and Kanamycin-Resistance in Transgenic Tobacco Plants

Inheritance of altered flower morphology and resistance to antibiotic kanamycin was studied in the first and second generations (T₁and T₂, respectively) of self-pollinated transgenic tobacco plants. In most transformants, kanamycin resistance was closely linked to mutant phenotype. T-DNA-induced mutations were shown to be dominant.     

Photosynthesis and Carbon Partitioning in Transgenic Tobacco Plants Deficient in Leaf Cytosolic Pyruvate Kinase

Whole-plant diurnal C exchange analysis provided a noninvasive estimation of daily net C gain in transgenic tobacco (Nicotiana tabacum L.) plants deficient in leaf cytosolic pyruvate kinase (PK_c−). PK_c− plants cultivated under a low light intensity (100 μmol m⁻² s⁻¹) were previously shown to exhibit markedly reduced root growth, as well as delayed shoot and flower development when compared with plants having wild-type levels of PK_c (PK_c+). PK_c− and PK_c+ source leaves showed a similar net C gain, photosynthesis over a range of light intensities, and a capacity to export newly fixed ¹⁴CO₂ during photosynthesis. However, during growth under low light the nighttime, export of previously fixed ¹⁴CO₂ by fully expanded PK_c− leaves was 40% lower, whereas concurrent respiratory ¹⁴CO₂ evolution was 40% higher than that of PK_c+ leaves. This provides a rationale for the reduced root growth of the PK_c− plants grown at low irradiance. Leaf photosynthetic and export characteristics in PK_c− and PK_c+ plants raised in a greenhouse during winter months resembled those of plants grown in chambers at low irradiance. The data suggest that PK_c in source leaves has a critical role in regulating nighttime respiration particularly when the available pool of photoassimilates for export and leaf respiratory processes are low.     

Restoration of Photosynthesis in Pot-Bound Tobacco Plants

Premature senescence is observed in pot-bound tobacco plants(Nicotiana tabacum L. cv. xanthii) and this is shown to be accompaniedby gross accumulation of leaf starch and a marked decline inphotosynthesis, ribulose- 1, 5-bisphosphate (RuBP) carboxylaseactivity, and soluble protein content. Starch content, uptakeof CO₂ by leaf discs, and the activity of RuBP carboxylase inwhole leaf extracts were measured daily following transfer ofrestricted plants to larger pots. Starch declined rapidly, whilstphotosynthetic activity was fully restored within a period ofseveral days, and frequently exceeded the maximum rates measuredin non-restricted plants. This overcompensation, which couldbe related to availability of carbohydrates accumulated as starch,is discussed in relation to the well-established horticulturalpractice of ‘potting on’.     

Peroxidase-Induced Wilting in Transgenic Tobacco Plants

Peroxidases are a family of isoenzymes found in all higher plants. However, little is known concerning their role in growth, development, or response to stress. Plant peroxidases are heme-containing monomeric glycoproteins that utilize either H2O2 or O2 to oxidize a wide variety of molecules. To obtain more information on possible in planta functions of peroxidases, we have used a cDNA clone for the primary isoenzyme form of peroxidase to synthesize high levels of this enzyme in transgenic plants. We were able to obtain Nicotiana tabacum and N. sylvestris transformed plants with peroxidase activity that is 10-fold higher than in wild-type plants by introducing a chimeric gene composed of the cauliflower mosaic virus 35S promoter and the tobacco anionic peroxidase cDNA. The elevated peroxidase activity was a result of increased levels of two anionic peroxidases in N. tabacum, which apparently differ in post-translational modification. Transformed plants of both species have the unique phenotype of chronic severe wilting through loss of turgor in leaves, which was initiated at the time of flowering. The peroxidase-induced wilting was shown not to be an effect of diminished water uptake through the roots, decreased conductance of water through the xylem, or increased water loss through the leaf surface or stomata. Possible explanations for the loss of turgor, and the significance of these types of experiments in studying isoenzyme families, are discussed.     

Transgenic Tobacco Plants Low in Phytochrome A Content

Tobacco (Nicotiana tabacum) plants were transformed with a construct encoding phytochrome A (PHYA) antisense RNA. The construct inserted into the tobacco genome contained squash PHYA cDNA in an antisense orientation under the cauliflower mosaic virus 35S promoter providing for gene expression in higher plant tissues. Using immunoblot analysis and Z3-B1 antibodies against PHYA, the authors demonstrated that the PHYA content of the transgenic plants was lower than that of the wild-type plants. The studies of PHYA-dependent inhibition of hypocotyl elongation by high-intensity far-red light showed a considerable decrease in light sensitivity of the transgenic hypocotyl characteristic for aphyAmutation.     

Photosynthesis in Salt-Adapted Heterotrophic Tobacco Cells and Regenerated Plants

Tobacco (Nicotiana tabacum L.) cells growing heterotrophically in the light on supplied sucrose (S0) have previously been adapted to grow in 428 mM NaCl (S25). Among the changes occurring in salinity-adapted cell cultures are (a) elevated levels of chlorophyll compared to unadapted cells; (b) decreased levels of starch; (c) alterations in chloroplast ultrastructure, including loss of starch grains, increased thylakoid membrane structure, and the presence of plastoglobules; and (d) increased rates of O2 evolution, CO2 fixation, and photophosphorylation relative to S0 cells. These latter changes apparently derive from the fact that thylakoid membranes in S25 cells contain higher levels of photosystem I- and II-associated proteins as well as thylakoid ATPase components. S25 chloroplasts contain immunologically detectable levels of ribulose-1,5-bisphosphate carboxylase/oxygenase, whereas S0 completely lack the enzyme. These changes taken together suggest that even in the presence of sucrose, S25 cells have acquired a significant degree of salt-tolerant photosynthetic competence. This salt-tolerant photoysynthetic capability manifests itself in plants backcrossed with normal plants for three generations. These plants contain chloroplasts that demonstrate in vitro more salt-tolerant CO2 fixation, O2 evolution, and photophosphorylation than do backcross progeny of plants regenerated from S0 cultures.     

Altered Epiphytic Colonization of Mannityl Opine-Producing Transgenic Tobacco Plants by a Mannityl Opine-Catabolizing Strain of Pseudomonas syringae

The plasmid pYDH208, which confers the ability to catabolize the mannityl opines mannopine and agropine, was mobilized into the nonpathogenic Pseudomonas syringae strain Cit7. The growth of the mannityl opine-catabolizing strain Cit7(pYDH208) was compared with that of the near-isogenic non-opine-catabolizing strain Cit7xylE on leaves of wild-type tobacco (Nicotiana tabacum cv. Xanthi) and transgenic mannityl opine-producing tobacco plants (N. tabacum cv. Xanthi, line 2-26). The population size of Cit7(pYDH208) was significantly greater on the lower leaves of transgenic plants than on middle or upper leaves of those plants. The population size of Cit7(pYDH208) on lower leaves of transgenic plants was also significantly greater than the population size of Cit7xylE on similar leaves of wild-type plants. High-voltage paper electrophoresis demonstrated higher levels of mannityl opines in washings from lower- and mid-level leaves than in washings from upper-level leaves. The ability of Cit7(pYDH208) to catabolize mannityl opines in the carbon-limited phyllosphere increased the carrying capacity of the lower leaves of transgenic plants for Cit7(pYDH208). In coinoculations, the increase in the ratio of population sizes of Cit7(pYDH208) to Cit7xylE on transgenic plants was apparently due to a subtle difference in the growth rates of the two strains and to the difference in final population sizes. An ability to utilize additional carbon sources on the transgenic plants also enabled Cit7(pYDH208) to achieve a higher degree of coexistence with Cit7xylE on transgenic plants than on wild-type plants. This supports the hypothesis that the level of coexistence between epiphytic bacterial populations can be altered through nutritional resource partitioning.     

Altered Growth of Transgenic Tobacco Lacking Leaf Cytosolic Pyruvate Kinase

Previously, we reported that transformation of tobacco (Nicotiana tabacum L.) with a vector containing a potato cytosolic pyruvate kinase (PK_c) cDNA generated two plant lines specifically lacking leaf PK_c (PK_c−) as a result of co-suppression. PK_c deficiency in these primary transformants did not appear to alter plant development, although root growth was not examined. Here we report a striking reduction in root growth of homozygous progeny of both PK_c− lines throughout development under moderate (600 μE m⁻² s⁻¹) or low (100 μE m⁻² s⁻¹) light intensities. When both PK_c− lines were cultivated under low light, shoot and flower development were also delayed and leaf indentations were apparent. Leaf PK activity in the transformants was significantly decreased at all time points examined, whereas root activities were unaffected. Polypeptides corresponding to PK_c were undetectable on immunoblots of PK_c− leaf extracts, except in 6-week-old low-light-grown PK_c− plants, in which leaf PK_c expression appeared to be greatly reduced. The metabolic implications of the kinetic characteristics of partially purified PK_c from wild-type tobacco leaves are discussed. Overall, the results suggest that leaf PK_c deficiency leads to a perturbation in source-sink relationships.     

两种凝集素基因在转基因烟草中表达的研究

构建了含尾穗苋凝集素基因(ACA)的cDNA序列和改造后的雪花莲凝集素基因(GNA)的植物表达载体pBACG。在此表达载体中,ACA和GNA基因的表达分别由35S启动子和CoYMV启动子控制。通过农杆菌介导,将ACA和GNA基因转化到烟草中,经卡那霉素筛选获得60株转化再生植株。对PCR检测呈阳性的50株植株进行接蚜虫实验,结果表明,其平均抑虫率达83．9％。Southern blotting分析表明,ACA和GNA基因都已整合到烟草基因组中。Western blotting结果显示这两个基因在不同植株中都可表达其相应的蛋白质,但表达水平不同。部分Western blotting分析呈阳性植株的抗蚜性与T0代相近,达85．3％,说明这两个基因的抗蚜功能可以稳定遗传。     

Expression of a Thermostable Bacterial Cellulase in Transgenic Tobacco Plants

The bacterial gene of the thermostable endo--1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased bushiness and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.     

Resistance Against Pepper Severe Mosaic Potyvirus in Transgenic Tobacco Plants

The coat protein gene of the pepper severe mosaic poty-virus was introduced into tobacco plants. Several transgenic lines were assayed for virus resistance under greenhouse conditions. Line 232, showing higher levels of resistance than other transgenic plants, was studied in further detail. Upon challenge with PepSMV, R₂ descendants from this line were monitored for virus accumulation for a period of 45 days. Virus titre was lower in plants of the line 232 than in control plants throughout the experiment. At 14 days post-inoculation the transgenic plant line reached the maximum virus accumulation measured by ELISA. Those plants also showed a 1 week delay of virus accumulation by comparison with control plants. After reaching the peak at 14 days post-inoculation the virus titre in line 232 decreased with respect to control plants, until the end of the experiment. These results are compatible with the presence of an RNA-mediated resistance mechanism.     

Transgenic Tobacco Plants Expressing Bacterial Genes for Thermostable Glucanases

It is shown that bacterial genes for thermostable -glucanases are expressed retaining their activity and substrate specificity. The leader peptide of the carrot extensin exerts effective secretion of the bacterial enzymes into the intercellular space of the plant tissue. Expression of the bacterial gene for -1,3-glucanase in plant tissues alters their morphogenetic potential. Regeneration of shoots from the calli of these plant lines requires a six- to eightfold increase in cytokinin (6-BAP) concentration in comparison with the control lines and the transgenic lines expressing -1,3-1,4-glucanase. Rooting of transgenic plants expressing the bacterial gene for -1,3-glucanase occurs much faster. The transgenic plants obtained in the study are proposed as model objects for investigating the role of glucanases in plants.     

Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO^M) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO^P) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO^P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO^P (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO^M (21%). The cytoprotective effect of the asialo-rhuEPO^P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.     

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism.