A highly selective alkaloid uptake system in vacuoles of higher plants

Vacuoles were isolated from different plant cell cultures and the transport mechanism for alkaloid uptake at the tonoplast membrane, as well as the compartmentation of enzymes and products inside the cells were investigated. While serpentine, the major alkaloid of Catharanthus roseus cells, is definitely located inside the vacuole, two key enzymes of the indole-alkaloid pathway, strictosidine synthase and a specific glucosidase, are located in the cytosol. Transport of alkaloids across the tonoplast into the vacuolar space has been characterized as an active, engergy-requiring mechanism, which is sensitive to the temperature and pH of the surrounding medium, stimulated by K⁺ and Mg²⁺, and inhibited by N,N-dicyclohexylcarbodiimid and Cu²⁺. The alkaloids accumulate inside the vacuoles against a concentration gradient, and the uptake system is specific for alkaloids indigenous to the plant from which the vacuoles have been isolated.Abbreviation DCCD N,N-dicyclohexylcarbodiimid Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Changes in phenolic metabolism of tobacco plants during short-term boron deficiency

The effects of boron (B) deficiency on several phenolics and enzyme activities involved in the biosynthesis of these compounds were investigated in tobacco plants (Nicotiana tabacum L. cv. Gatersleben). The levels of phenylpropanoids (mainly the caffeic acid esters, chlorogenic acid and its isomers) as well as phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and polyphenoloxidase (PPO, EC 1.14.18.1) activities were determined in plants subjected to B starvation for 1–7 d. The results presented here show that a short-term B deficiency causes both quantitative and qualitative changes in the phenolic metabolism of tobacco plants, which are especially evident after 3 d of B starvation. Although the concentration of B decreased from the onset of B starvation, root B level was less affected than leaf B by a short-term B deficiency. The concentration of phenylpropanoids as well as PAL and PPO activities increased mainly in the leaves of tobacco plants during B starvation. Moreover, leaves starved of B for 7 d showed the accumulation of new compounds, one of which was identified as caffeoylputrescine. In addition, a positive correlation between PAL activity and phenylpropanoid concentration was observed in tobacco leaves, especially after 5–7 d of B starvation, suggesting that an increase in PAL activity during B starvation could be responsible for the enhancement in the levels of phenylpropanoids.     

Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants

Strictosidine, a precursor to over 1000 indole alkaloids including the anti-tumor drugs vinblastine, vincristine, and camptothecin, is produced by the condensation of tryptamine and secologanin. Strictosidine synthase, the enzyme responsible for this condensation, is the first committed step in the indole-alkaloid pathway. We have introduced a modified cDNA encoding Strictosidine synthase from Catharanthus roseus (L.) Don. (McKnight et al. 1990, Nucl. Acids Res. 18, 4939) driven by the CaMV 35S promoter into tobacco (Nicotiana tabacum L.). Transgenic tobacco plants expressing this construct had from 3 to 22 times greater strictosidinesynthase activity than C. roseus plants. Ultrastructural immunolocalization demonstrated that strictosidine synthase is a vacuolar protein in C. roseus and is correctly targeted to the vacuole in transgenic tobacco. Immunoblot analysis of strictosidine synthase showed that two distinct forms of the enzyme were produced in transgenic tobacco plants but that only a single form was made in C. roseus. This observation indicates that the second form of the protein is not simply a result of overexpression in tobacco, but may reflect differences in protein processing between tobacco and C. roseus.Abbreviations cDNA complementary DNA - TLC thin-layer chromatography We thank Dr. C.A. Roessner for providing the E. coli strain expressing strictosidine synthase, Dr. J. Balsevich for providing alkaloid standards, and Dr. L. Cloney for assisting with antibody preparation. This work was supported by a National Institutes of Health Biomedical Research Support Grant to T.D.M and by a grant from the US Department of Agriculture, Competitive Research Grants Office (90-37262-5375) to C.L.N.     

Analysis of insecticidal activity in transgenic plants carrying the ipt plant growth hormone gene

The expression of a bacterial cytokinin biosynthesis gene (PI-II-ipt) in Nicotiana plumbaginifolia Viviani plants has been correlated with enhanced resistance to Manduca sexta and Myzus persicae. We expressed the PI-II-ipt gene in N. tabacum and Lycopersicon esculentum and observed similar antifeedent effects with the transgenic tobacco but not tomato. A 30 to 50 % reduction in larval weight gain was observed with some of the tomato plants but these results could not be repeated consistently. Leaf surface extracts from transgenic N. plumbaginifolia leaves killed 100 % of M. sexta second instars at concentrations of 0.05 % (w/v) whereas the N. tabacum extracts were at least 20 times less active. Extract suspensions were stable for up to 2 days at ambient temperatures below 42 °C and for at least 3 months at 4 °C when stored in the dark. HPLC analysis of the N. plumbaginifolia extracts yielded an active fraction that reduced hatching of M. sexta eggs by 30 % and killed first, second and third instars within 24, 48 and 72 hours of exposure, respectively. The activity appears to be associated with oxygen-containing aliphatic compounds, possibly diterpenes, as analyzed by TLC, UV absorption and fragmentation with EIMS. Based on the partial characterization of this activity, the production, secretion or accumulation of secondary metabolites in leaves of cytokinin producing PI-II-ipt N. plumbagini-folia plants appears to be responsible for the observed insect resistance.     

Use of a simple semiquantitative method for appraisal of green fluorescent protein gene expression in transgenic tobacco plants

We have applied a simple method for evaluation of gfp gene expression in plants using a CCD camera and computerized processing of images. Transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transfer of plasmid T-DNA bearing a m-gfp5-ER sequence governed by the 35S promoter together with the nptII selectable marker gene. Presence of the gfp gene in plants was confirmed by a polymerase chain reaction method. Mean brightness values measured using image analysis software showed differences between transgenic and control plants and suggest the possibility of rapid selection of transgenic individuals among regenerants and their progenies.     

Effects of ammonia on carbon metabolism in photosynthesizing isolated mesophyll cells from Papaver somniferum L.

Addition of ammonia to a suspension of photosynthesizing isolated mesophyll cells from P. somniferum quantitatively alters the pattern of carbon metabolism by increasing rates of certain key ratelimiting steps leading to amino-acid synthesis and by decreasing rates of rate-limiting steps in alternative biosynthetic pathways. Of particular importance is the stimulation of reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase. The increased rates of these two reactions, which result in an increased flow of carbon into the tricarboxylic-acid cycle, correlate with a rapid rise in glutamine (via glutamine synthetase) which draws carbon off the tricarboxylic-acid cycle as -ketoglutarate. Increased flux of carbon in this direction appears to come mainly at the expense of sucrose synthesis. The net effect of addition of ammonia to mesophyll cells is thus a redistribution of newly fixed carbon away from carbohydrates and into amino acids.     

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose 6-phosphate - FW fresh weight - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - 3PGA glycerate-3-phosphate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - Pi inorganic phosphate - PPi inorganic pyrophosphate - UDPGlc UDP-glucose This research was supported by the Deutsche Forschungsgemein-Schaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister für Forschung und Technologie (U.S., L.W.).     

Evaluation of acute and chronic toxicity of selected compounds in higher plants using cell culture

Summary The feasibility of using plant cell culture to measure toxicity was determined by investigating the toxicological effects of three chemical compounds, allyl alcohol, propargylglycine, and cadmium chloride, on cell cultures ofCatharanthus roseus G. Don (Madagascar periwinkle). Suspension cultures ofC. roseus were maintained in modified B5 medium and transferred every 5 d. Five-day-old cell cultures were exposed to various concentrations (10,3,1,0.3,0.1,0.03,0.01,0.003,0.001,0.0003,0.0001, 0.00003, and 0.0 mM) of the toxicants in both acute and chronic toxicity tests. In the acute test, cells were exposed to the toxicant for 24 h, washed three times with sterile medium, and plated in petri plates with an equal volume of 1.4% agar medium. Cells in the chronic test were plated with an equal volume of 1.4% agar medium containing various concentrations of the toxicant. Cells were incubated 28 d at 30°C in the dark. The colonies were counted and the results plotted as percent survival versus toxicant concentration. The results indicate, at the concentrations tested, thatC. roseus assay may be feasible in that it fulfills the criteria for a practical assay (e.g., rapid, simple, quantifiable, and reproducible). This work was submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Environmental Science, Institute of Environmental Sciences.     

Transformation of Actinidia eriantha: A potential species for functional genomics studies in Actinidia

Protocols were developed for regeneration and Agrobacterium-mediated transformation of Actinidia eriantha Benth. A. eriantha has a number of features that make it a useful tool for functional genomics in Actinidia: the vines are relatively small and non-vigorous in nature, flowers form all over the vine including on lower axillary branches and the species flowers prolifically in greenhouse conditions. Flowering and fruiting of transgenic A. eriantha plants was obtained within 2 years of transformation in a containment greenhouse. GUS (β-glucuronidase) activity indicating stable expression of the uidA gene was observed in leaf, stem, root, petal and fruit tissues. Molecular evidence for incorporation of transgenes into the A. eriantha genome was obtained by PCR and DNA gel blot analysis. Inheritance of transgenic phenotypes was demonstrated in seedling progeny. Functional genomic studies in kiwifruit have been initiated using transgenic A. eriantha plants. Communicated by F. Sato     

Developmental changes of antioxidative systems in tobacco leaves as affected by limited sucrose export in transgenic plants expressing yeast-invertase in the apoplastic space

It is generally believed that a restricted export of carbohydrates from source leaves causes oxidative stress because of an enhanced utilisation of O₂ instead of NADP⁺ as electron acceptor in photosynthesis. To test this hypothesis, developmental changes of antioxidative systems were investigated in wild-type and transgenic tobacco (Nicotiana tabacum L.) suffering from disturbed sink-source relations by expression of yeast invertase in the apoplastic space. Young expanding leaves of the wild type contained higher activities of Superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), glutathione reductase (EC 1.6.4.2) and a higher glutathione content than mature source leaves. The activity of monodehydroascorbate-radical reductase (EC 1.1.5.4) and the ascorbate content remained unaffected by the developmental stage in the wild type. In young expanding leaves of the transgenic plants the capacity of the antioxidative systems was similar to or higher than in corresponding leaves from the wild type. Source leaves of transgenic tobacco with an increased carbohydrate content showed a small chlorophyll loss, an increased malondialdehyde content, a selective loss of the activities of Cu/Zn-superoxide dismutase isoenzymes and a fourfold decrease in ascorbate compared with the wild type. There was no evidence that the protection from H₂O₂ was insufficient since source leaves of transgenic tobacco contained increased activities of catalase, ascorbate peroxidase, and monodehydroascorbate-radical reductase and an increased ascorbate-to-dehydroascorbate ratio compared with source leaves of the wild type. In severely chlorotic leaf sections of the transgenic plants, most components of the antioxidative system were lower than in green leaf sections, but the ascorbate-to-dehydroascorbate ratio was increased. These results suggest that carbohydrate-accumulating cells have an increased availability of reductant, which can increase the degree of reduction of the ascorbate system via glutathione-related systems or via the activity of monodehydroascorbate-radical reductase. At the same time, transgenic tobacco leaves seem to suffer from an increased oxidative stress, presumably as a result of a decreased consumption of O ₂ ^.- by Cu/Zn-superoxide dismutases in the chloroplasts. There was no evidence that carbohydrate-accumulating leaves acclimated to enhanced O ₂ ^.- production rates in the chloroplasts.     

Transgenic tobacco plants producing caffeine: a potential new strategy for insect pest control

Caffeine (1,3,7–trimethylxanthine) is one of the most widely used plant secondary metabolites, primarily as a stimulant and an ingredient in drugs. In nature, caffeine is believed to function in chemical defense, acting as an antiherbivory and allelopathic agent, and therefore it might be employed to protect agriculturally important crop plants. In coffee plants, caffeine is synthesized from the precursor xanthosine in four steps, three N-methylations and removal of ribose. We had previously isolated genes encoding three distinct N-methyltransferases, and we demonstrated production of recombinant enzymes that yielded caffeine in in vitro reconstitution experiments. When these caffeine biosynthetic pathway genes were simultaneously expressed in tobacco plants (Nicotiana tabacum), caffeine was successfully produced up to 5 μg/g fresh weight in leaves. The leaves were unpalatable to tobacco cutworms (Spodoptera litura). This repellent action appeared to be more widely␣applicable to lepidopteran caterpillars as observed with small white (Pieris rapae) fed on Chinese cabbages that had been top-treated with caffeine. Our recent results suggest a novel approach to strengthen anti-herbivore traits by producing caffeine in crop plants.