Potato hexokinase 2 complements transgenic Arabidopsis plants deficient in hexokinase 1 but does not play a key role in tuber carbohydrate metabolism

Potato plants (Solanum tuberosum L. cv. Désirée) transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 2 exhibited altered enzyme activities and expression of hexokinase 2 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed an 11-fold variation in leaf (from 48% of the wild-type activity in antisense transformants to 446% activity in sense transformants) and an 8-fold variation in developing tubers (from 35% of the wild-type activity in antisense transformants to 212% activity in sense transformants). Despite the wide range of hexokinase activities, no substantial change was found in the fresh weight yield, starch, sugar and metabolite levels of transgenic tubers. However, both potato hexokinases 1 and 2 were able to complement the hyposensitivity of antisense hexokinase 1 Arabidopsis transgenic plants to glucose. In an in vitro bioassay of seed germination in a medium with high glucose levels, double transformants showed the same sensitivity to glucose as that of the wild-type ecotype, displaying a stunted phenotype in hypocotyls, cotyledons and roots.     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

The effect of chimeric transgene architecture on co-ordinated gene silencing

Two gene constructs were made consisting of a 244-bp sense fragment from the 5′ end of a polygalacturonase cDNA, the 3′ end of which was ligated to a 414-bp fragment from the 5′ end of a phytoene synthase cDNA. In the first construct, the phytoene synthase fragment was in a sense orientation (sense/sense chimeric gene) and in the second construct the phytoene synthase fragment was in an antisense orientation (sense/antisense chimeric gene). Both chimeric genes were inserted between a cauliflower mosaic virus promoter and terminator. Tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) plants transformed with each construct gave rise to transformants with three distinct phenotypes: plants with red fruit, plants with pure yellow fruit and plants with red and yellow sectored fruit. For both chimeric constructs, expression of the endogenous polygalacturonase and phytoene synthase genes were found to be co-ordinately suppressed in yellow tissue, but showed normal expression in red tissue. Data from microscopic analyses of fruit chromoplasts, from the three phenotypes, implied that phytoene synthase suppression from each construct predominantly had two states within a cell: on or off. Received: 31 July 1997 / Accepted: 28 August 1997     

Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress

Glycinebetaine is one of the compatible solutes that accumulate in the chloroplasts of certain halotolerant plants when these plants are exposed to salt or cold stress. The codA gene for choline oxidase, the enzyme that converts choline into glycinebetaine, has previously been cloned from a soil bacterium, Arthrobacter globiformis. Transformation of Arabidopsis thaliana with the cloned codA gene under the control of the 35S promoter of cauliflower mosaic virus enabled the plant to accumulate glycinebetaine and enhanced its tolerance to salt and cold stress. At 300 mM NaCl, considerable proportions of seeds of transformed plants germinated well, whereas seeds of wild-type plants failed to germinate. At 100 mM NaCl, transformed plants grew well whereas wild-type plants did not do so. The transformed plants tolerated 200 mM NaCl, which was lethal to wild-type plants. After plants had been incubated with 400 mM NaCl for two days, the photosystem II activity of wild-type plants had almost completely disappeared, whereas that of transformed plants remained at more than 50% of the original level. When exposed to a low temperature in the light, leaves of wild-type plants exhibited symptoms of chlorosis, whereas those of transformed plants did not. These observations demonstrate that the genetic modification of Arabidopsis thaliana that allowed it to accumulate glycinebetaine enhanced its ability to tolerate salt and cold stress.     

Normal starch content and composition in tubers of antisense potato plants lacking D-enzyme (4-α-glucanotransferase)

Transgenic potato (Solanum tuberosum L.) plants were created with sense and antisense copies of the potato D-enzyme (disproportionating enzyme; EC␣2.4.1.25) cDNA linked to patatin and cauliflower mosaic virus 35 S promoters, and screened for D-enzyme activity in tubers. Transformants with sense constructs mostly had wild type D-enzyme activity but two plants had only about 1% wild-type activity. Transformants with antisense constructs had activity ranging from 90% to about 1% of wild type. Three 35 S antisense plants with very low activity were analysed in detail. Western blot analysis showed that D-enzyme was present in greatly reduced amounts in tubers and in leaves, whereas plastidic starch phosphorylase (EC 2.4.1.1) was unaffected. The lack of D-enzyme resulted in slow plant growth but development was otherwise apparently normal. Furthermore, the starch content of tubers was not appreciably altered in amount, proportion of amylose, molecular weight of debranched amylopectin, or branch chain length, despite the lack of D-enzyme. These results do not indicate a direct requirement for D-enzyme in the synthesis and accumulation of storage starch in tubers. The results are discussed in terms of the known reactions catalysed by D-enzyme and possible involvement of D-enzyme in starch metabolism. Received: 12 November 1997 / Accepted: 23 December 1997     

Abnormal `wrinkled' cell walls and retarded development of transgenic Arabidopsis thaliana plants expressing endo-1,4-β-glucanase (cell) antisense

Transgenic Arabidopsis thaliana plants expressing cell antisense exhibit reduced levels of cell mRNA and protein compared with wild-type plants. The former display significant alterations in their phenotype. cell antisense plants have shorter stems and roots and are mechanically weaker than their wild-type counterparts. In cell antisense plants, the cell wall structure is markedly disrupted: both fluorescent confocal microscopy and scanning electron microscopy revealed `wrinkled' cell walls, thus indicating that CEL1 plays an important role in cell wall relaxation during cell growth and expansion. In cell antisense plants, the number of xylem elements per bundle is smaller than in the wild-type. In addition, both xylem elements and interfascicular fibers are significantly less lignified in the former. It is suggested that in A. thaliana, abnormal cell wall deposition affected by CEL1 depletion is associated not only with cell growth, but also with the differentiation process in the vascular and supporting tissues.Equal contributors     

Enhancing the Anaerobic Response

Proteome analysis, and more recently DNA chip technology, hasled to the identification of a large number of genes that areimplicated in the anaerobic response of plants. As a result,an increasingly complex picture of the response in terms ofbiochemical and regulatory processes is emerging. A challengeis to find out more about the function of these newly identifiedgene products, and how they contribute to flooding tolerance.Our approach has been to manipulate levels of candidate geneproducts (using sense and antisense constructs) in the modelsystem Arabidopsis thaliana, combined with biochemical and phenotypicanalysis of the resulting transgenic plants.     

Strategies for the suppression of peroxidase gene expression in tobacco. II.In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs

Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expressionin planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the ligninforming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40–80% was seen in the T₀ and T₁ populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzsin vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.     

Subcellular and tissue localization of NAD kinases from Arabidopsis: compartmentalization of de novo NADP biosynthesis

The de novo biosynthesis of the triphosphopyridine NADP is catalyzed solely by the ubiquitous NAD kinase family. The Arabidopsis (Arabidopsis thaliana) genome contains two genes encoding NAD⁺ kinases (NADKs), annotated as NADK1, NADK2, and one gene encoding a NADH kinase, NADK3, the latter isoform preferring NADH as a substrate. Here, we examined the tissue-specific and developmental expression patterns of the three NADKs using transgenic plants stably transformed with NADK promoter::glucuronidase (GUS) reporter gene constructs. We observed distinct spatial and temporal patterns of GUS activity among the NADK::GUS plants. All three NADK::GUS transgenes were expressed in reproductive tissue, whereas NADK1::GUS activity was found mainly in the roots, NADK2::GUS in leaves, and NADK3::GUS was restricted primarily to leaf vasculature and lateral root primordia. We also examined the subcellular distribution of the three NADK isoforms using NADK–green fluorescent protein (GFP) fusion proteins expressed transiently in Arabidopsis suspension-cultured cells. NADK1 and NADK2 were found to be localized to the cytosol and plastid stroma, respectively, consistent with previous work, whereas NADK3 localized to the peroxisomal matrix via a novel type 1 peroxisomal targeting signal. The specific subcellular and tissue distribution profiles among the three NADK isoforms and their possible non-overlapping roles in NADP(H) biosynthesis in plant cells are discussed.     

Loss of decreased-rubisco phenotype between generations of wheat transformed with antisense and sense rbcS

The elite UK winter wheat cv. Riband was transformed with constructs containing rbcS in sense and antisense orientations driven by the maize ubiquitin promoter with a transformation efficiency of 1.2%. Of 77 primary transformants 31% of the sense-rbcS transformed lines and 78% of the antisense-rbcS transformed lines had decreased rubisco content compared to wild-type and marker-only controls, with decreases of up to 60%. However, in the T1 progeny which inherited the transgene, only 5% showed significantly decreased rubisco content and these effects were on the margins of significance. Five potential T2 homozygous lines from T1 parents which had transgene segregation consistent with a single locus were identified. There was no significant decrease in rubisco content relative to wild-type in any of these lines (LSD of 8% for P= 0.05). Expression of antisense rbcS transgenes in two of these T2 lines was low but was increased following exposure of the plants to 37°C for 48 h. However this did not induce a significant decrease in rubisco protein content relative to controls. Southern analysis of two antisense lines showed that they had low copy number and 1–2 insertion events. In one of the two lines there was increased methylation of the ubiquitin intron in T2 samples compared to the TO primary transformant. Further work is required to establish whether methylation occurred in all the lines which lost the phenotype, and therefore the likelihood of this being the cause. The disappearance of the decreased rubisco-content phenotype between generations may therefore be attributable to (1) greater activity of the ubiquitin promoter due to greater stress in the T0 generation plants and/or (2) increased methylation of the transgene promoter region between generations.     

Simultaneous suppression of three genes related to brassinosteroid (BR) biosynthesis altered campesterol and BR contents, and led to a dwarf phenotype in Arabidopsis thaliana

We generated transgenic lines of Arabidopsis thaliana with an RNA interference construct that expressed hairpin double-stranded RNA for DET2:DWF4:SMT2 to induce sequence-specific RNA silencing. In transgenic plants, expressions of DET2, DWF4, and SMT2 were simultaneously reduced, and the campesterol content was increased by up to 420% compared to the level in the wild-type plant. Triple knock-down of the DET2, DWF4, and SMT2 enzymes also resulted in reduction of brassinosteroid (BR)-specific biosynthesis intermediates. Transgenic plants harboring the RNA interference construct displayed a semi-dwarf phenotype due to altered development. Our findings indicate that redesigning of plant architecture is possible through simultaneous suppression of multiple genes involved in BR biosynthesis.     

Control of potato tuber dormancy and sprouting by expression of sense and antisense genes of pyrophosphatase in potato

Inorganic pyrophosphate (PPi) is an enzyme involved in sugar metabolism in potato tubers. In our previous study, we isolated an inorganic pyrophosphatase (PPase) gene from potato and obtained the transgenic potato plants transformed with the sense and antisense PPase genes respectively. In the present experiment, the physiological indexes, tuber dormancy, and sprouting characteristics of the transgenic potatoes were analyzed and evaluated. The result showed that the PPase activity and the inorganic phosphate content of tubers were lower in the antisense transgenic plant lines but were higher in the sense transgenic plant lines, compared with wild-type tubers. Soluble sugars, such as glucose, fructose and sucrose increased in transgenic plants that had overexpression of the sense PPase gene, but decreased in the antisense transgenic plant lines, compared with wild-type tubers. Tuber sprouting time of the antisense transgenic plants were delayed for 2 and 3 weeks and reached the 100 % sprouting rate only after 14 and 16 weeks storage compared with the wild-type when tubers are stored under 25 and 4 °C, respectively. In contrast, tuber sprouting time of the sense transgenic plants was earlier by approximately 2 weeks than that of wild-type tubers under these storage temperatures.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

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     

Expression of Helicobacter pylori TonB protein in transgenic Arabidopsis thaliana: toward production of vaccine antigens in plants

Background: The aim of this study was to produce a recombinant version of the highly antigenic Helicobacter pylori TonB (iron‐dependent siderophore transporter protein HP1341) in transgenic plants as a candidate oral vaccine antigen. Materials and Methods: Using Agrobacterium‐mediated gene transfer, we introduced three different constructs of the tonB gene into the genome of the model plant Arabidopsis thaliana. We investigated transgene insertion by PCR, produced TonB antibodies for analysis of the production of the recombinant protein in plants, verified the identity of the protein produced by mass spectrometry analysis, and analyzed the number of genetic inserts in the plants by Southern blotting. Results: Three different constructs of the expression cassette (full‐length tonB, tonB truncated in the 5′ end removing the codons for a transmembrane helix, and the latter construct with codons for the endoplasmic reticulum SEKDEL retention signal added to the 3′ end) were used to find the most effective way to express the TonB antigen. Production of TonB protein was detected in plants transformed with each of the constructs, confirmed by both Western blotting and mass spectrometry analysis. No considerable differences in protein expression from the three different constructs were observed. The protein concentration in the plants was at least 0.05% of the total soluble proteins. Conclusions: The Helicobacter pylori TonB protein can be produced in Arabidopsis thaliana plants in a form that is recognizable by rabbit anti‐TonB antiserum. These TonB‐expressing plants are highly suitable for animal studies of oral adminstration as a route for immunization against Helicobacter infections.     

The production of an inducible antisense alternative oxidase (Aox1a) plant

Plant mitochondria contain an alternative oxidase (AOX) acting as a terminal electron acceptor of the alternative pathway in the electron transport chain. Here we describe the production of inducible antisense Aox1a plants of Arabidopsis thaliana (L.) Heynh. and the procedures used to determine the resulting alternative pathway activity. The Arabidopsis Aox1a cDNA sequence was cloned behind a copper-inducible promoter system in the antisense orientation. Arabidopsis thaliana (Columbia) plants were transformed by in-planta vacuum infiltration with Agrobacterium containing the antisense construct. Whole-leaf ethanol production was used as a measure to investigate alternative pathway activity in the presence of antimycin A. After 24 h, leaves from the copper-induced, antisense line F1.1 produced up to 8.8 times more ethanol (via aerobic fermentation) than the non-induced and wild-type leaves, indicating effective cytochrome pathway inhibition by antimycin A and a decreased alternative pathway activity in induced F1.1 leaves. Transgene expression studies also revealed no expression in non-induced leaves and up until 24 h post-induction. Copper-induced transgenic leaves were less susceptible to alternative pathway inhibition than non-induced transgenic leaves, as seen via tissue-slice respiratory studies, and mitochondrial respiration, using F1.1 cell cultures, also supported this. These results demonstrate the successful production of a transgenic line of Arabidopsis in which the alternative pathway activity can be genetically manipulated with an inducible antisense system. Received: 31 March 2000 / Accepted: 10 May 2000