Expression of antibodies and Fab fragments in transgenic potato plants: a case study for bulk production in crop plants

To use transgenic potato tubers (Solanum tuberosum cv. Désirée) for bulk production of recombinant antibodies, constructs were engineered for accumulating full-size IgGs and Fab fragments in the plant cell apoplast or endoplasmic reticulum (ER). An in-house transformation protocol was worked out for the efficient co-transformation of potato root explants. Accumulation levels in tubers of up to 0.5% of total soluble protein were found for antibodies targeted to the ER whereas five-fold lower accumulation levels were found for antibodies targeted for secretion. Additionally, different aspects important for the commercial exploitation of potato tubers as a heterologous production system were analysed. Tubers could be stored for up to 6 months without significant loss of antibody amount or activity. Minor variations in antibody accumulation levels were observed in tubers that originated from the same transformant. Most isolated IgGs and Fab fragments bound the antigen and had the correct molecular weight when compared with the hybridoma-derived standard. Processing to greenhouse or field trials, including in vitro propagation of a selected transformant, required only approximately 9 months from the start of transformation, a time frame in which hundreds of kilograms of transgenic potato tubers could easily be obtained. Small-scale purification of IgG was possible by using standard laboratory techniques. Thus, molecular farming in potato tubers can be a viable production system for economic production of clinically or industrially interesting macromolecules, such as antibodies.     

Expression of the Galanthus nivalis agglutinin (GNA) gene in transgenic potato plants confers resistance to aphids

Aphids, the largest group of sap-sucking pests, cause significant yield losses in agricultural crops worldwide every year. The massive use of pesticides to combat this pest causes severe damage to the environment, putting in risk the human health. In this study, transgenic potato plants expressing Galanthus nivalis agglutinin (GNA) gene were developed using CaMV 35S and ST-LS1 promoters generating six transgenic lines (35S1-35S3 and ST1-ST3 corresponding to the first and second promoter, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the GNA gene was expressed in leaves, stems and roots of transgenic plants under the control of the CaMV 35S promoter, while it was only expressed in leaves and stems under the control of the ST-LS1 promoter. The levels of aphid mortality after 5 days of the inoculation in the assessed transgenic lines ranged from 20 to 53.3%. The range of the aphid population in transgenic plants 15 days after inoculation was between 17.0 ± 1.43 (ST2) and 36.6 ± 0.99 (35S3) aphids per plant, which corresponds to 24.9–53.5% of the aphid population in non-transformed plants. The results of our study suggest that GNA expressed in transgenic potato plants confers a potential tolerance to aphid attack, which appears to be an alternative against the use of pesticides in the future.     

Fusion proteins comprising the catalytic domain of mutansucrase and a starch-binding domain can alter the morphology of amylose-free potato starch granules during biosynthesis

It has been shown previously that mutan can be co-synthesized with starch when a truncated mutansucrase (GtfICAT) is directed to potato tuber amyloplasts. The mutan seemed to adhere to the isolated starch granules, but it was not incorporated in the starch granules. In this study, GtfICAT was fused to the N- or C-terminus of a starch-binding domain (SBD). These constructs were introduced into two genetically different potato backgrounds (cv. Kardal and amf), in order to bring GtfICAT in more intimate contact with growing starch granules, and to facilitate the incorporation of mutan polymers in starch. Fusion proteins of the appropriate size were evidenced in starch granules, particularly in the amf background. The starches from the various GtfICAT/SBD transformants seemed to contain less mutan than those from transformants with GtfICAT alone, suggesting that the appended SBD might inhibit the activity of GtfICAT in the engineered fusion proteins. Scanning electron microscopy showed that expression of SBD-GtfICAT resulted in alterations of granule morphology in both genetic backgrounds. Surprisingly, the amf starches containing SBD-GtfICAT had a spongeous appearance, i.e., the granule surface contained many small holes and grooves, suggesting that this fusion protein can interfere with the lateral interactions of amylopectin sidechains. No differences in physico-chemical properties of the transgenic starches were observed. Our results show that expression of granule-bound and “soluble” GtfICAT can affect starch biosynthesis differently.     

Expression of the potato leafroll luteovirus coat protein gene in transgenic potato plants inhibits viral infection

Transgenic potato plants, cultivar Désirée, were produced that contained the coat protein gene of potato leafroll luteovirus (PLRV). The transformed potato plants expressed the PLRV coat protein (CP) RNA sequences but accumulation of coat protein in transgenic tissues could not be detected. Upon inoculation with PLRV, the PLRV CP RNA expressing potato plants showed a reduced rate of virus multiplication.     

Expression of full-length bioactive antimicrobial human lactoferrin in potato plants

A cDNA fragment encoding human lactoferrin (hLF) linked to a plant microsomal retention signal peptide (SEKDEL) was stably integrated into the Solanum tuberosum genome by Agrobacterium tumefaciens-mediated leaf disk transformation methods. The lactoferrin gene was expressed under control of both the auxin-inducible manopine synthase (mas) P2 promoter and the cauliflower mosaic virus (CaMV) 35S tandem promoter. The presence of the hLF cDNA in the genome of regenerated transformed potato plants was detected by polymerase chain reaction amplification methods. Full-length hLF protein was identified by immunoblot analysis in tuber tissue extracts from the transformed plants by immunoblot analysis. The hLF produced in transgenic plant tissues migrated during polyacrylamide gel electrophoresis as a single band with an approximate molecular mass equal to hLF. Auxin activation of the mas P2 promoter increased lactoferrin expression levels in transformed tuber and leaf tissues to approximately 0.1% of total soluble plant protein. Antimicrobial activity against four different human pathogenic bacterial strains was detected in extracts of lactoferrin-containing potato tuber tissues. This is the first report of synthesis of full length, biologically active hLF in edible plants.     

Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355

The gene encoding alternansucrase (ASR) from Leuconostoc mesenteroides NRRL B-1355, an original sucrose glucosyltransferase (GTF) specific to alternating alpha-1,3 and alpha-1,6 glucosidic bond synthesis, was cloned, sequenced and expressed into Escherichia coli. Recombinant enzyme catalyzed oligoalternan synthesis from sucrose and maltose acceptor. From sequence comparison, it appears that ASR possesses the same domains as those described for GTFs specific to either contiguous alpha-1,3 osidic bond or contiguous alpha-1,6 osidic bond synthesis. However, the variable region and the glucan binding domain are longer than in other GTFs (by 100 and 200 amino acids respectively). The N-catalytic domain which presents 49% identity with the other GTFs from L. mesenteroides possesses the three determinants potentially involved in the glucosyl enzyme formation.     

Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. For the example of maltose as the acceptor, if one considers only the linear, unbranched possibilities for alternansucrase, the hypothetical number of potential products increases exponentially as a function of the degree of polymerization (DP). Experimental evidence indicates that far fewer products are actually formed. We show that only certain isomers of DP >4 are formed from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. When the oligosaccharide acceptor products from maltose were separated by size-exclusion chromatography and HPLC, only one pentasaccharide was isolated. Its structure was alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Two hexasaccharides were formed in approximately equal quantities: alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc and alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Just one heptasaccharide was isolated from the reaction mixture, alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. We conclude that the enzyme is incapable of forming two consecutive alpha-(1-->3) linkages, and does not form products with more than two consecutive alpha-(1-->6) linkages. The distribution of products may be kinetically determined.     

Sucrose synthase activity does not restrict glycolysis in roots of transgenic potato plants under hypoxic conditions

The effect of hypoxia on root development and carbon metabolism was studied using potato (Solanum tuberosum L.) plants as a model system. Hypoxia led to a cessation of root elongation, and finally to the death of meristematic cells. These changes were accompanied by a 4- to 5-fold accumulation of hexoses, suggesting that insufficient carbohydrate supply was not the cause of cell death. In addition, prolonged hypoxia (96 h) resulted in a 50% increase in activity of most glycolytic enzymes studied and the accumulation of glycerate-3-phosphate and phosphoenolpyruvate. This indicates that endproduct utilisation may restrict metabolic flux through glycolysis. As expected, the activities of alcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.17) increased during hypoxia. Apart from the enzymes of ethanolic fermentation the activity of sucrose synthase (SuSy; EC 2.4.1.13) was enhanced. To investigate the in-vivo significance of this increase, transgenic plants with reduced SuSy activity were analysed. Compared to untransformed controls, transgenic plants showed a reduced ability to resume growth after re-aeration, emphasising the crucial role of SuSy in the toleration of hypoxia. Surprisingly, analysis of glycolytic intermediates in root extracts from SuSy antisense plants revealed no change as compared to wildtype plants. Therefore, limitation of glycolysis is most likely not responsible for the observed decreased ability for recovery after prolonged oxygen starvation. We assume that the function of SuSy during hypoxia might be to channel excess carbohydrates into cell wall polymers for later consumption rather than fuelling glycolysis. Received: 17 February 1999 / Accepted: 10 June 1999     

Expression of neutralizing epitope of porcine epidemic diarrhea virus in potato plants

Transgenic plants expressing recombinant proteins from pathogenic microorganisms provide an inexpensive edible vaccine for induction of local immunity. A neutralizing epitope of porcine epidemic diarrhea virus (PEDV) gene containing SEKDEL was expressed in potato using Agrobacterium-mediated transformation system. Putative transgenic plants were regenerated, and genomic PCR confirmed the presence of PEDV epitope gene in the potato plants. Based on the ELISA results, epitope of PEDV protein made up approximately 0.1% of the total soluble tuber protein.     

Expression of cholera toxin B subunit oligomers in transgenic potato plants

A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (Mr 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (Mr 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world     

Antisense inhibition of cytosolic NADP-dependent isocitrate dehydrogenase in transgenic potato plants

Cytosolic NADP-dependent isocitrate dehydrogenase (cyt-NADP-ICDH; EC 1.1.1.42) has been suggested to play a major role in the production of 2-oxoglutarate, an important precursor for amino acid synthesis. Using an antisense RNA approach under the control of the cauliflower mosaic virus 35S promoter, transgenic potato plants were created in which NADP-ICDH activity was reduced to 8% of the wild-type level in leaves. Residual activity was almost completely due to mitochondrial and chloroplastic NADP-ICDH isoforms. Activity staining after non-denaturing polyacrylamide gel electrophoresis revealed the complete absence of a major activity band in leaves of antisense plants. No differences in growth or development, including flower formation and tuber yield, were observed between transgenic and wild-type plants. Photosynthesis and respiration were also unchanged. Levels of amino acids were the same in wild-type and cyt-NADP-ICDH antisense plants, even when accumulation of amino acids was induced by incubation of detached leaves in tap water in the dark (`induced senescence'). Consistent with a reduction in NADP-ICDH activity, however, were slight increases in the levels of isocitrate (up to 2.5-fold) and citrate (up to 2-fold). 2-Oxoglutarate was not reduced. Our data indicate that potato plants can cope with a severe reduction in cyt-NADP-ICDH activity without major shifts in growth and metabolism. Received: 28 July 1997 / Accepted: 3 November 1997     

Acceptor products of alternansucrase with gentiobiose. Production of novel oligosaccharides for food and feed and elimination of bitterness

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. Previously, we showed that alternansucrase only forms certain isomers of DP > 4 from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. We now demonstrate that the acceptor products from gentiobiose, also formed in good yields (nearly 90% in unoptimized reactions), follow a pattern similar to those formed from maltose. The initial product is a single trisaccharide, α-d-Glcp-(1→6)-β-d-Glcp-(1→6)-d-Glc. Two tetrasaccharides were formed in approximately equal quantities: α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-β-d-Glcp-(1→6)-d-Glc and α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-β-d-Glcp-(1→6)-d-Glc. Just one pentasaccharide was isolated from the reaction mixture, α-d-Glcp-(1→6)-α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-β-d-Glcp-(1→6)-d-Glc. Our hypothesis that the enzyme is incapable of forming two consecutive α-(1→3) linkages, and does not form products with more than two consecutive α-(1→6) linkages, apparently applies to other acceptors as well as to maltose. The glucosylation of gentiobiose reduces or eliminates its bitter taste.     

Production of dextran in transgenic potato plants

The production of dextran in potato tubers and its effect on starch biosynthesis were investigated. The mature dextransucrase (DsrS) gene from Leuconostoc mesenteroides was fused to the chloroplastic ferredoxin signal peptide (FD) enabling amyloplast entry, which was driven by the highly tuber-expressed patatin promoter. After transformation of two potato genotypes (cv. Kardal and the amylose-free (amf) mutant), dextrans were detected by enzyme-linked immunosorbent assay (ELISA) in tuber juices of Kardal and amf transformants. The dextran concentration appeared two times higher in the Kardal (about 1.7 mg/g FW) than in the amf transformants. No dextran was detected by ELISA inside the starch granule. Interestingly, starch granule morphology was affected, which might be explained by the accumulation of dextran in tuber juices. In spite of that, no significant changes of the physicochemical properties of the starches were detected. Furthermore, we have observed no clear changes in chain length distributions, despite the known high acceptor efficiency of DSRS.     

Metabolic response of potato plants to an antisense reduction of the P-protein of glycine decarboxylase

Potato (Solanum tuberosum L. cv. Desiré) plants with reduced amounts of P-protein, one of the subunits of glycine decarboxylase (GDC), have been generated by introduction of an antisense transgene. Two transgenic lines, containing about 60–70% less P-protein in the leaves compared to wild-type potato, were analysed in more detail. The reduction in P-protein amount led to a decrease in the ability of leaf mitochondria to decarboxylate glycine. Photosynthetic and growth rates were reduced but the plants were viable under ambient air and produced tubers. Glycine concentrations within the leaves were elevated up to about 100-fold during illumination. Effects on other amino acids and on sucrose and hexoses were minor. Nearly all of the glycine accumulated during the day was metabolised during the following night. The data suggest that the GDC operates far below substrate saturation under normal conditions thus allowing a flexible and fast response to changes in the environment. Received: 4 March 2000 / Accepted: 26 July 2000     

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     

Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol

Potato (Solanum tuberosum cv. Désirée) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised. All lines of transgenic plants showed similarities to plants growing under water stress. Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types. In all transformants rates of CO₂ assimilation and leaf conductance were reduced. From the unchanged intercellular partial pressure of CO₂ and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO₂ assimilation was not caused by a limitation in the availability of CO₂ for␣the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered. In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates. But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical. Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants. Received: 29 May 1996 / Accepted: 30 December 1996     

Inducible cross-tolerance to herbicides in transgenic potato plants with the rat CYP1A1 gene

A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 μmol/pot), whereas no accumulation of the corresponding mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1 cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides. These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred chemical-inducible herbicide tolerance on potato. Received: 15 March 2001 / Accepted: 14 June 2001     

Karyotypic changes in potato plants regenerated from protoplasts

Over two hundred plants were regenerated from shoot-culture derived proto-plasts of potato (Solanum tuberosum L. cv. Majestic). Some had grossly aberrant phenotypes but the majority were similar to, or indistinguishable from normal control Majestic. Cytological examination showed that on average, 57% of the regenerants had the normal chromosome number (2n=4x=48). The remainder were aneuploids and fell into two classes in approximately equal numbers. The first class was limited at about the euploid level (ie, 2n=44–49). The second class contained plants with higher chromosome numbers ranging from 2n=73 to the octaploid level (2n=8x=96). The overall results represent an improvement over our earlier studies on chromosome variation in protoplast-derived potato plants. In addition, three cases of structural chromosome variation were observed.     

Construction and functional characteristics of tuber-specific and cold-inducible chimeric promoters in potato

The improvement of processing quality of potato products (fries and chips) demands less accumulation of reducing sugars (glucose and fructose) in cold-stored potato (Solanum tuberosum) tubers. Control of gene expression to achieve this requires promoters with specificity to tubers as well as inducible activity under low temperatures. Here we use overlapping extension PCR to construct two chimeric promoters, pCL and pLC, to control gene expression in a tuber-specific and cold-inducible pattern. This combined different combinations of the LTRE (low-temperature responsive element) from Arabidopsis thaliana cor15a promoter and the TSSR (tuber-specific and sucrose-responsive sequence) from potato class I patatin promoter. The cold-inducible and tuber-specific activities of the chimeric promoters were investigated by quantitative analysis of GUS activity in transgenic potato cultivar E3 plants. The results showed that the cis-elements, LTRE and TSSR, played responsive roles individually or in combination. pCL with the TSSR closer to the TATA-box showed substantially higher promoter activity than pLC with the LTRE closer to the TATA-box at either normal (20°C) or low temperature (2°C), suggesting that the promoter activity was closely associated with the position of the two elements. The chimeric promoter pCL with tuber-specific and cold-inducible features may provide valuable tool for controlling the expression of gene constructs designed to lower the formation of reducing sugars in tubers stored at low temperature and to improve the processing quality of potato products. The nucleotide sequence data reported will appear in the GenBank database under the accession numbers DQ494557 (pCL) and DQ494558 (pLC ).