Kiwifruits (Actinidia deliciosa) transformed with a Vitis stilbene synthase gene produce piceid (resveratrol-glucoside)

A stilbene synthase gene was isolated from three Vitis spp. (V. vinifera, V. labrusca and V. riparia). These genes were placed under the control of the cauliflower mosaic virus 35S promoter and introduced into kiwifruit (Actinidia deliciosa) plants by Agrobacterium-mediated gene transfer. The introduced gene(s) were expressed and piceid (resveratrol-glucoside) rather than resveratrol was produced in the leaves of the transformants. Resveratrol produced by the action of the integrated gene(s) seems to be metabolized into piceid by an endogenous glycosyltransferase. Among the transformants obtained, the highest piceid content in the young leaves was 182 μg/g fresh weight. Although these transformants did not show resistance against Botrytis cinerea, which causes gray-mould disease, the fruits may have some beneficial effects on human health. Received: 2 September 1999 / Revision received: 10 December 1999 / Accepted: 19 December 1999     

Drought induces fructan synthesis and 1-SST (sucrose: sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.)

 Seeds of Cichorium intybus L. var. foliosum cv. Flash were sown in acid-washed vermiculite and grown in a controlled-environment growth chamber. After 1 month of growth, plantlets did not contain sucrose:sucrose 1-fructosyltransferase (1-SST), the key enzyme in fructan biosynthesis. No fructan could be observed. Some of the plants were submitted to drought for 2 weeks. Glucose, fructose and sucrose concentrations increased in roots and leaves of stressed plants and the fructan concentration in roots and leaves was ten times higher than in control plants. The onset of fructan synthesis coincided with the increase in 1-SST activity in roots. Expression of the 1-SST gene could be observed in roots and leaves of stressed plants. Received: 12 July 1999 / Accepted: 16 October 1999     

Transgenic potato plants with altered expression levels of chloroplast NADP-malate dehydrogenase: interactions between photosynthetic electron transport and malate metabolism in leaves and in isolated intact chloroplasts

The contribution of the malate valve in the regulation of steady-state photosynthesis was studied in transgenic potato (Solanum tuberosum L. cv Désirée) plants with altered expression of plastidic NADP-dependent malate dehydrogenase (NADP-MDH; EC 1.1.1.82). Mutant plants were obtained after transformation with the homologous Nmdh gene in antisense orientation, or with the Nmdh gene from pea (Pisum sativum L.) in sense orientation. A total number of nine stable sense and antisense lines with 10% or 30%, and 400% of wild-type NADP-MDH capacity were selected. Intact chloroplasts were isolated from leaves of wild-type and mutant plants. In chloroplasts from sense transformants the increased enzyme amount was activated as in wild-type chloroplasts, but increased rates of oxaloacetate-dependent malate formation were only measured upon partial uncoupling. In contrast, chloroplasts from antisense transformants produced only little malate upon oxaloacetate addition. Measurements with intact leaves during steady-state photosynthesis yielded no differences in gas-exchange parameters and chlorophyll fluorescence. The leaf malate content was unchanged in NADP-MDH underexpressors, but twice as high in overexpressing plants. The altered NADP-MDH expression clearly influences the redox state of ferredoxin, especially in low light. Furthermore, the malate valve can successfully compete for electrons with cyclic electron flow, but the conditions under which this occurs are quite artificial. Received: 14 February 1998 / Accepted: 12 May 1998     

The isolation and characterisation of a mutant of the C4 plant Amaranthus edulis deficient in NAD-malic enzyme activity

A mutant of Amaranthus edulis (Speg.) lacking activity of the C₄ leaf form of NAD-malic enzyme (ME; EC 1.1.1.39) has been isolated. Homozygous mutant (5% wild-type ME activity) and heterozygous (50% wild-type ME activity) F₂ plants were shown to contain both the α and β NAD-ME subunits in similar amounts to those detected in the wild-type leaves. The rate of photosynthetic CO₂ assimilation was reduced in the homozygous mutant to 5% of that observed for the wild-type leaves. Other C₄ enzymes were not down-regulated in the mutant plants. There was little difference in photosynthetic rate of the heterozygous plants compared to the wild-type, suggesting that NAD-ME exerts little control over the rate of C₄ photosynthesis, and that in the wild-type the enzyme has a very low control coefficient. The activity loss in the heterozygote may therefore be compensated by regulatory mechanisms that increase the activity of the enzyme in vivo. Data for bundle-sheath strands indicated that although the homozygous mutants were able to oxidise malate via the Krebs cycle, they were unable to convert malate to pyruvate and alanine via NAD-ME. Received: 2 April 1998 / Accepted: 7 May 1998     

Tyrosinase involved in betalain biosynthesis of higher plants

A tyrosine-hydroxylating enzyme was partially purified from betacyanin-producing callus cultures of Portulaca grandiflora Hook. by using hydroxyapatite chromatography and gel filtration. It was characterized as a tyrosinase (EC 1.14.18.1 and EC 1.10.3.1) by inhibition experiments with copper-chelating agents and detection of concomitant o-diphenol oxidase activity. The tyrosinase catalysed both the formation of L-(3,4-dihydroxyphenyl)-alanine (Dopa) and cyclo-Dopa which are the pivotal precursors in betalain biosynthesis. The hydroxylating activity with a pH optimum of 5.7 was specific for L-tyrosine and exhibited reaction velocities with L-tyrosine and D-tyrosine in a ratio of 1:0.2. Other monophenolic substrates tested were not accepted. The enzyme appeared to be a monomer with an apparent molecular mass of ca. 53 kDa as estimated by gel filtration and SDS-PAGE. Some other betalain-producing plants and cell cultures were screened for tyrosinase activity; however, activities could only be detected in red callus cultures and plants of P. grandiflora as well as in plants, hairy roots and cell cultures of Beta vulgaris L. subsp. vulgaris (Garden Beet Group), showing a clear correlation between enzyme activity and betacyanin content in young B. vulgaris plants. We propose that this tyrosinase is specifically involved in the betalain biosynthesis of higher plants. Received: 14 July 1998 / Accepted: 23 October 1998     

Biochemical and molecular characterization of transgenic<Emphasis Type="Italic"> Lotus japonicus</Emphasis> plants constitutively over-expressing a cytosolic glutamine synthetase gene

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS₁) or in the chloroplast (GS₂). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS₁ gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS₁ polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.Abbreviations GS Glutamine synthetase - UTR Untranslated region     

Ultrastructural effects in potato leaves due to antisense-inhibition of the sucrose transporter indicate an apoplasmic mode of phloem loading

To study the export of sugars from leaves and their long-distance transport, sucrose-proton/co-transporter activity of potato was inhibited by antisense repression of StSUT1 under control of either a ubiquitously active (CaMV 35S ) or a companion-cell-specific (rolC) promotor in transgenic plants. Transformants exhibiting reduced levels of the sucrose-transporter mRNA and showing a dramatic reduction in root and tuber growth, were chosen to investigate the ultrastructure of their source leaves. The transformants had a regular leaf anatomy with a single-layered palisade parenchyma, and bicollateral minor veins within the spongy parenchyma. Regardless of the promoter used, source leaves from transformants showed an altered leaf phenotype and a permanent accumulation of assimilates as indicated by the number and size of starch grains, and by the occurrence of lipid-storing oleosomes. Starch accumulated throughout the leaf: in epidermis, mesophyll and, to a smaller degree, in phloem parenchyma cells of minor veins. Oleosomes were observed equally in mesophyll and phloem parenchyma cells. Companion cells were not involved in lipid accmulation and their chloroplasts developed only small starch grains. The similarity of ultrastructural symptoms under both promotors corresponds to, rather than contradicts, the hypothesis that assimilates can move symplasmically from mesophyll, via the bundle sheath, up to the phloem. The microscopical symptoms of a constitutively high sugar level in the transformant leaves were compared with those in wild-type plants after cold-girdling of the petiole. Inhibition of sugar export, both by a reduction of sucrose carriers in the sieve element/companion cell complex (se/cc complex), or further downstream by cold-girdling, equally evokes the accumulation of assimilates in all leaf tissues up to the se/cc complex border. However, microscopy revealed that antisense inhibition of loading produces a persistently high sugar level throughout the leaf, while cold-girdling leads only to local patches containing high levels of sugar. Received: 4 March 1998 / Accepted: 7 April 1998     

In vivo and in vitro flowering response of chicory (Cichorium intybus L.): influence of plant age and vernalization

Chicory plants (Cichorium intybus L. var foliosum cv Flash) were tested with and without a 4-week-long cold treatment for in vivo and in vitro flowering potential every 2 weeks during the growing season. One hundred percent of the plants harvested 112 days or later after sowing and then vernalized flowered in vivo. In vitro, no vernalization was needed to initiate flowering-stems on chicory explants taken from roots of 100 days old and older. 5-Azacytidine, a DNA demethylation agent, increased the flowering percentage on explants from young, vernalized roots but could not induce more than 15% flowering on young, nonvernalized roots. The greater flowering potential of chicory root explants in vitro when compared to plants of the same age tested in vivo was clearly established. This result suggests that some negative control on flowering was removed when root explants were excised and the main plant body discarded. Received: 31 August 1998 / Revision received: 27 October 1998 / Accepted: 10 November 1998     

Morphological changes in transgenic poplar induced by expression of the rice homeobox gene OSH1

Genetically transformed lombardy poplar (Po-pulus nigra L. var. italica Koehne) plants were regenerated after co-cultivation of stem segments with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector which included the rice gene for a homeodomain protein (OSH1) and a gene for neomycin phosphotransferase. The expression of the OSH1 gene under control of the cauliflower mosaic virus 35S promoter induced morphological abnormalities in the leaves and stems of the newly generated transgenic poplar plants. This result suggests that OSH1 can function as a regulator of morphogenesis in transgenic poplar, as it does in transgenic rice, Arabidopsis, and tobacco plants. Received: 16 October 1998 / Revision received: 27 November 1998 / Accepted: 12 December 1998     

Transformation of lisianthus (Eustoma grandiflorum)

Transformed plants from three cultivars of Eustoma grandiflorum (lisianthus) were produced by cocultivating young leaf pieces with Agrobacterium tumefaciens strain A722 containing the binary vectors pKIWI110 and pLN26. Both vectors contain the selectable marker gene for neomycin phosphotransferase II. pKIWI110 also contains the reporter gene for β-d-glucuronidase, and pLN26, the chalcone synthase antisense gene. Southern DNA analysis revealed that all the kanamycin-resistant transformants tested contained copies of the transgenes integrated in their genome. The two plants transformed with pKIWI110 show β-d-glucuronidase expression in their mature leaves and selected transformants passed on the kanamycin-resistant phenotype to the F1 generation. Received: 8 January 1997 / Revision received: 12 May 1997 / Accepted: 3 June 1997     

Transformation of arctic bramble (Rubus arcticus L.) by Agrobacterium tumefaciens

Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which β-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited with cefotaxime. Kanamycin was used as the selective agent for the transformants. Regenerants were assayed by histochemical GUS staining, and by Southern analysis using a gus-int probe. Transgenic arctic bramble plants containing gus-int and expressing GUS were recovered. Expression has been stable for 3 years in micropropagation. Received: 22 October 1997 / Revision received: 7 January 1998 / Accepted: 2 February 1998     

Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion

Plant ageing and senescence are associated with increased levels of reactive oxygen species. Level of cytokinins, the apparent inhibitors of plant senescence, is controlled by their irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). We investigated the CKX activity, cytokinin concentration, and activities of antioxidative enzymes in tobacco (Nicotiana tabacum L. cv. Samsun NN) overexpressing the Arabidopsis gene for AtCKX2, targeted for extracellular secretion pathway. The control and AtCKX2 plants differed substantially in their phenotypes. When the lowest leaves in controls became yellow all leaves in AtCKX2 tobacco still remained green. Activities of antioxidant enzymes decreased with leaf age in both tobacco plants except for ascorbate peroxidase (APX) in the old leaves and glutathione reductase (GR) in young leaves. Enhancement of GR activity at all leaf stages, an increase of superoxide dismutase and a decline of catalase in young leaves, as well as an increase of APX in the oldest leaves were observed in AtCKX2 plant compared to control. Similar changes were detected after determination of isoenzymes on zymograms. It is evident that AtCKX2 plants had postponed onset of senescence despite the significantly lowered level of cytokinins. Enhanced antioxidant protection, especially in the oldest leaves, could subsidise this phenomenon.     

Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry

A new method for the selection of transgenic plants has been developed. It is based upon selection of transgenic plant cells expressing the xylA gene from Streptomyces rubiginosus, which encodes xylose isomerase, on medium containing xylose. The xylose isomerase selection system was tested in potato and the transformation frequency was found to be approximately ten fold higher than with kanamycin selection. The level of enzyme activity in the transgenic plants selected on xylose was 5- to 25-fold higher than the enzyme activity in control plants. Potato transformants were stable over two generations in Southern blotting analysis. This novel selection system is more efficient than the traditionally used kanamycin-based selection systems. In addition, the xylose isomerase system is independent of antibiotic or herbicide resistance genes, but depends on an enzyme that is generally recognized as safe for use in the starch industry and which is already being widely utilized in specific food processes. Received: 13 August 1997 / Revision received: 26 November 1997 / Accepted: 15 December 1997     

Resistance to Alternaria brassicicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene

Transgenic broccoli plants expressing a Trichoderma harzianum endochitinase gene were obtained by Agrobacterium tumefaciens-mediated transformation. PCR and Southern blot analysis confirmed the presence of the gene in plants initially selected via resistance to kanamycin. Primary transformants (T₀) and selfed progeny (T₁) were examined for expression of the endochitinase gene using a fluorometric assay and for their resistance to the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum. All transgenic plants with elevated endochitinase activity had the expected 42 kDa endochitinase band in western blot analysis, whereas no such band was detected in the non-transgenic control. Leaves of most mature T₀ plants had 14–37 times higher endochitinase activity than controls; mature T₁ plants had higher endochitinase activity (100–200 times that in controls), in part because of lower control values. T₀ plantlets in vitro or young plants in soil had higher absolute and relative endochitinase activity. When detached leaves of T₀ plants were inoculated with A. brassicicola, lesion size showed a significant negative correlation with endochitinase levels. After inoculation of two-month old T₀ plants with A. brassicicola, all 15 transgenic lines tested showed significantly less severe disease symptoms than controls. In contrast, lesion size on petioles of T₀ and T₁ plants inoculated with S. sclerotiorum was not statistically different from controls.     

Effect of chitinase antisense RNA expression on disease susceptibility of Arabidopsis plants

Chitinases accumulate in higher plants upon pathogen attack are capable of hydrolyzing chitin-containing fungal cell walls and are thus implicated as part of the plant defense response to fungal pathogens. To evaluate the relative role of the predominate chitinase (class I, basic enzyme) of Arabidopsis thaliana in disease resistance, transgenic Arabidopsis plants were generated that expressed antisense RNA to the class I chitinase. Young plants or young leaves of some plants expressing antisense RNA had <10% of the chitinase levels of control plants. In the oldest leaves of these antisense plants, chitinase levels rose to 37–90% of the chitinase levels relative to vector control plants, most likely because of accumulation and storage of the enzyme in vacuoles. The rate of infection by the fungal pathogen Botrytis cinerea was measured in detached leaves containing 7–15% of the chitinase levels of control plants prior to inoculation. Antisense RNA was not effective in suppressing induced chitinase expression upon infection as chitinase levels increased in antisense leaves to 47% of levels in control leaves within 24 hours after inoculation. Leaves from antisense plants became diseased at a slightly faster rate than leaves from control plants, but differences were not significant due to high variability. Although the tendency to increased susceptibility in antisense plants suggests that chitinases may slow the growth of invading fungal pathogens, the overall contribution of chitinase to the inducible defense reponses in Arabidopsis remains unclear.     

Overexpression of a cyanobacterial phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase with diminished sensitivity to feedback inhibition in<Emphasis Type="Italic"> Arabidopsis</Emphasis> changes amino acid metabolism

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, l-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C₄-like pathway is proposed.Abbreviations CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kan Kanamycin - 2-ME 2-Mercaptoethanol - MS/G medium 1/2 Murashige–Skoog and 1/2 Gamborg mixed medium - PEP Phosphoenolpyruvate - PEPC Phosphoenolpyruvate carboxylase - Sv Synechococcus vulcanus - ZmPEPC Maize PEPC involved in C₄ photosynthesis     

Electrotransformation of Chlorella vulgaris

Using hygromycin B resistance as a marker for selection, we have established the conditions required for the transformation of Chlorella vulgaris. The exponentially grown C. vulgaris cells were transformed by electroporation with plasmid pIG121-Hm, and transformants were selected with hygromycin B at a concentration of 50 μg/ml. Cell extracts prepared from the late-log cultures of the transformants exhibited glucuronidase activities as conferred by the gus gene on pIG121-Hm. The maintenance of plasmid in the algal cells seemed to be transient as many cultures derived from the hygromycin B-resistant colonies gradually lost the hygromycin resistance upon prolonged growth. The result of Southern blotting of the genomic DNAs prepared from transformant cultures exhibiting persistent hygromycin resistance showed that integration of part of the plasmid DNA into the host chromosome had taken place. Received: 19 December 1997 / Revision received: 5 October 1998 / Accepted: 27 October 1998