The genetic and functional analysis of flavor in commercial tomato : the FLORAL4 gene underlies a QTL for floral aroma volatiles in tomato fruit
Tikunov, Yury M. ; Roohanitaziani, Raana ; Meijer-Dekens, Fien ; Molthoff, Jos ; Paulo, Joao ; Finkers, Richard ; Capel, Iris ; Carvajal Moreno, Fatima ; Maliepaard, Chris ; Nijenhuis-de Vries, Mariska ; Labrie, Caroline W. ; Verkerke, Wouter ; Heusden, Adriaan W. van; Eeuwijk, Fred van; Visser, Richard G.F. ; Bovy, Arnaud G. - \ 2020
The Plant Journal (2020). - ISSN 0960-7412
2-phenylethanol - aroma - flavor - quantitative trait loci - Solanum lycopersicum - tomato - volatiles
Tomato (Solanum lycopersicum L.) has become a popular model for genetic studies of fruit flavor in the last two decades. In this article we present a study of tomato fruit flavor, including an analysis of the genetic, metabolic and sensorial variation of a collection of contemporary commercial glasshouse tomato cultivars, followed by a validation of the associations found by quantitative trait locus (QTL) analysis of representative biparental segregating populations. This led to the identification of the major sensorial and chemical components determining fruit flavor variation and detection of the underlying QTLs. The high representation of QTL haplotypes in the breeders’ germplasm suggests that there is great potential for applying these QTLs in current breeding programs aimed at improving tomato flavor. A QTL on chromosome 4 was found to affect the levels of the phenylalanine-derived volatiles (PHEVs) 2-phenylethanol, phenylacetaldehyde and 1-nitro-2-phenylethane. Fruits of near-isogenic lines contrasting for this locus and in the composition of PHEVs significantly differed in the perception of fruity and rose-hip-like aroma. The PHEV locus was fine mapped, which allowed for the identification of FLORAL4 as a candidate gene for PHEV regulation. Using a gene-editing-based (CRISPR-CAS9) reverse-genetics approach, FLORAL4 was demonstrated to be the key factor in this QTL affecting PHEV accumulation in tomato fruit.
Identification of loci affecting accumulation of secondary metabolites in tomato fruit of a Solanum lycopersicum × Solanum chmielewskii introgression line population
Ballester Frutos, A.R. ; Tikunov, Yury ; Molthoff, Jos ; Grandillo, Silvana ; Viquez-Zamora, Marcela ; Vos, Ric de; Maagd, Ruud A. de; Heusden, Sjaak van; Bovy, Arnaud G. - \ 2016
Frontiers in Plant Science 7 (2016). - ISSN 1664-462X
Alkaloids - Flavonoids - Introgression lines - QTL analysis - Tomato (Solanum lycopersicum)
Semi-polar metabolites such as flavonoids, phenolic acids, and alkaloids are very important health-related compounds in tomato. As a first step to identify genes responsible for the synthesis of semi-polar metabolites, quantitative trait loci (QTLs) that influence the semi-polar metabolite content in red-ripe tomato fruit were identified, by characterizing fruits of a population of introgression lines (ILs) derived from a cross between the cultivated tomato Solanum lycopersicum and the wild species Solanum chmielewskii. By analyzing fruits of plants grown at two different locations, we were able to identify robust metabolite QTLs for changes in phenylpropanoid glycoconjugation on chromosome 9, for accumulation of flavonol glycosides on chromosome 5, and for alkaloids on chromosome 7. To further characterize the QTLs we used a combination of genome sequencing, transcriptomics and targeted metabolomics to identify candidate key genes underlying the observed metabolic variation.
Genetic mapping of semi-polar metabolites in pepper fruits (Capsicum sp.): towards unravelling the molecular regulation of flavonoid quantitative trait loci
Wahyuni, Y. ; Stahl-Hermes, V. ; Ballester, A.R. ; Vos, C.H. de; Voorrips, R.E. ; Maharijaya, A. ; Molthoff, J.W. ; Víquez Zamora, A.M. ; Sudarmonowati, E. ; Arisi, A.C.M. ; Bino, R.J. ; Bovy, A.G. - \ 2014
Molecular Breeding 33 (2014)3. - ISSN 1380-3743 - p. 503 - 518.
l. var. acuminatum - tomato fruit - frankliniella-occidentalis - capsaicinoid content - mass-spectrometry - annuum - expression - metabolomics - glycosides - biodiversity
Untargeted LCMS profiling of semi-polar metabolites followed by metabolite quantitative trait locus (mQTL) analysis was performed in ripe pepper fruits of 113 F2 plants derived from a cross between Capsicum annuum AC1979 (no. 19) and Capsicum chinense No. 4661 Selection (no. 18). The parental accessions were selected based on their variation in fruit morphological characteristics and fruit content of some target phytonutrients. Clear segregation of fruit colour and fruit metabolite profiles was observed in the F2 population. The F2 plants formed three clusters based on their metabolite profiles. Of the total of 542 metabolites, 52 could be annotated, including a range of flavonoids, such as flavone C-glycosides, flavonol O-glycosides and naringenin chalcone, as well as several phenylpropanoids, a capsaicin analogue, fatty acid derivatives and amino acid derivatives. Interval mapping revealed 279 mQTLs in total. Two mQTL hotspots were found on chromosome 9. These two chromosomal regions regulated the relative levels of 35 and 103 metabolites, respectively. Analysis also revealed an mQTL for a capsaicin analogue, located on chromosome 7. Confirmation of flavonoid mQTLs using a set of six flavonoid candidate gene markers and their corresponding expression data (expression QTLs) indicated the Ca-MYB12 transcription factor gene on chromosome 1 and the gene encoding flavone synthase (FS-2) on chromosome 6 as likely causative genes determining the variation in naringenin chalcone and flavone C-glycosides, respectively, in this population. The combination of large-scale metabolite profiling and QTL analysis provided valuable insight into the genomic regions and genes important for the production of (secondary) metabolites in pepper fruit. This will impact breeding strategies aimed at optimising the content of specific metabolites in pepper fruit
NON-SMOKY GLYCOSYLTRANSFERASE1 Prevents the Release of Smoky Aroma from Tomato Fruit
Tikunov, Y.M. ; Molthoff, J.W. ; Vos, R.C.H. de; Beekwilder, M.J. ; Houwelingen, A.M.M.L. van; Hooft, J.J.J. van der; Nijenhuis-de Vries, M.A. ; Labrie, C.W. ; Verkerke, W. ; Geest, H.C. van de; Víquez Zamora, A.M. ; Presa, S. ; Rambla Nebot, J.L. ; Granell, A. ; Hall, R.D. ; Bovy, A.G. - \ 2013
The Plant Cell 25 (2013)8. - ISSN 1040-4651 - p. 3067 - 3078.
mass spectrometry - small molecules - salicylic-acid - key enzyme - flavor - volatiles - biosynthesis - components - odor - gene
Phenylpropanoid volatiles are responsible for the key tomato fruit (Solanum lycopersicum) aroma attribute termed “smoky.” Release of these volatiles from their glycosylated precursors, rather than their biosynthesis, is the major determinant of smoky aroma in cultivated tomato. Using a combinatorial omics approach, we identified the NON-SMOKY GLYCOSYLTRANSFERASE1 (NSGT1) gene. Expression of NSGT1 is induced during fruit ripening, and the encoded enzyme converts the cleavable diglycosides of the smoky-related phenylpropanoid volatiles into noncleavable triglycosides, thereby preventing their deglycosylation and release from tomato fruit upon tissue disruption. In an nsgt1/nsgt1 background, further glycosylation of phenylpropanoid volatile diglycosides does not occur, thereby enabling their cleavage and the release of corresponding volatiles. Using reverse genetics approaches, the NSGT1-mediated glycosylation was shown to be the molecular mechanism underlying the major quantitative trait locus for smoky aroma. Sensory trials with transgenic fruits, in which the inactive nsgt1 was complemented with the functional NSGT1, showed a significant and perceivable reduction in smoky aroma. NSGT1 may be used in a precision breeding strategy toward development of tomato fruits with distinct flavor phenotypes.
Biochemical and molecular analysis of pink tomatoes: deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit colour
Ballester, A.R. ; Molthoff, J.W. ; Vos, C.H. de; Lintel Hekkert, B. ; Orzaez, D. ; Fernandez-Moreno, J.P. ; Tripodi, S. ; Grandillo, S. ; Martin, C. ; Heldens, J. ; Ykema, M. ; Granell, A. ; Bovy, A.G. - \ 2010
Plant Physiology 152 (2010). - ISSN 0032-0889 - p. 71 - 84.
arabidopsis-thaliana - flavonoid biosynthesis - in-vivo - anthocyanin - carotenoids - health - accumulation - inheritance - metabolome - mutations
The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink- colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.
Transcription analysis of apple fruit development using cDNA microarrays
Soglio, V. ; Costa, F. ; Molthoff, J.W. ; Weemen-Hendriks, M. ; Schouten, H.J. ; Gianfranceschi, L. - \ 2009
Tree Genetics and Genomes 5 (2009)4. - ISSN 1614-2942 - p. 685 - 698.
expressed-sequence-tags - beta-cyanoalanine synthase - malus-pumila mill. - gene-expression - ethylene biosynthesis - domestica borkh. - tomato - model - identification - maturation
The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and maturation thus potentially involved in fruit quality traits. The expression profile of 1,536 transcripts was analysed by microarray hybridisation. A total of 177 genes resulted to be differentially expressed in at least one of the developmental stages considered. Gene ontology annotation was employed to univocally describe gene function, while cluster analysis allowed grouping genes according to their expression profile. An overview of the transcriptional changes and of the metabolic pathways involved in fruit development was obtained. As expected, August and September are the two months where the largest number of differentially expressed genes was observed. In particular, 85 genes resulted to be up-regulated in September. Even though most of the differentially expressed genes are involved in primary metabolism, several other interesting functions were detected and will be presented.
Induction of lipid oxidation by polyunsaturated fatty acids of marine origin in small intestine of mice fed a high-fat diet
Schothorst, E.M. van; Flachs, P. ; Franssen-Hal, N.L.W. van; Kuda, O. ; Bunschoten, A. ; Molthoff, J.W. ; Vink, C. ; Hooiveld, G.J.E.J. ; Kopecky, J. ; Keijer, J. - \ 2009
BMC Genomics 10 (2009). - ISSN 1471-2164 - 11 p.
body-weight gain - gene-expression - beta-oxidation - c57bl/6j mice - fish-oil - inflammatory processes - obesity-resistance - adipose-tissue - omega-3 fats - metabolism
Background. Dietary polyunsaturated fatty acids (PUFA), in particular the long chain marine fatty acids docosahexaenoic (DHA) and eicosapentaenoic (EPA), are linked to many health benefits in humans and in animal models. Little is known of the molecular response to DHA and EPA of the small intestine, and the potential contribution of this organ to the beneficial effects of these fatty acids. Here, we assessed gene expression changes induced by DHA and EPA in the wildtype C57BL/6J murine small intestine using whole genome microarrays and functionally characterized the most prominent biological process. Results. The main biological process affected based on gene expression analysis was lipid metabolism. Fatty acid uptake, peroxisomal and mitochondrial beta-oxidation, and omega-oxidation of fatty acids were all increased. Quantitative real time PCR, and -in a second animal experiment- intestinal fatty acid oxidation measurements confirmed significant gene expression differences and showed in a dose-dependent manner significant changes at biological functional level. Furthermore, no major changes in the expression of lipid metabolism genes were observed in the colon. Conclusion. We show that marine n-3 fatty acids regulate small intestinal gene expression and increase fatty acid oxidation. Since this organ contributes significantly to whole organism energy use, this effect on the small intestine may well contribute to the beneficial physiological effects of marine PUFAs under conditions that will normally lead to development of obesity, insulin resistance and diabetes
The Impact of the Absence of Aliphatic Glucosinolates on Insect Herbivory in Arabidopsis
Beekwilder, J. ; Leeuwen, W. van; Dam, N.M. van; Bertossi, M. ; Grandi, V. ; Mizzi, L. ; Soloview, M. ; Szabados, L. ; Molthoff, J.W. ; Schipper, B. ; Verbocht, H. ; Vos, C.H. de; Morandini, P. ; Aarts, M.G.M. ; Bovy, A.G. - \ 2008
PLoS ONE 3 (2008)4. - ISSN 1932-6203 - 12 p.
Aliphatic glucosinolates are compounds which occur in high concentrations in Arabidopsis thaliana and other Brassicaceae species. They are important for the resistance of the plant to pest insects. Previously, the biosynthesis of these compounds was shown to be regulated by transcription factors MYB28 and MYB29. We now show that MYB28 and MYB29 are partially redundant, but in the absence of both, the synthesis of all aliphatic glucosinolates is blocked. Untargeted and targeted biochemical analyses of leaf metabolites showed that differences between single and double knock-out mutants and wild type plants were restricted to glucosinolates. Biosynthesis of long-chain aliphatic glucosinolates was blocked by the myb28 mutation, while short-chain aliphatic glucosinolates were reduced by about 50% in both the myb28 and the myb29 single mutants. Most remarkably, all aliphatic glucosinolates were completely absent in the double mutant. Expression of glucosinolate biosynthetic genes was slightly but significantly reduced by the single myb mutations, while the double mutation resulted in a drastic decrease in expression of these genes. Since the myb28myb29 double mutant is the first Arabidopsis genotype without any aliphatic glucosinolates, we used it to establish the relevance of aliphatic glucosinolate biosynthesis to herbivory by larvae of the lepidopteran insect Mamestra brassicae. Plant damage correlated inversely to the levels of aliphatic glucosinolates observed in those plants: Larval weight gain was 2.6 fold higher on the double myb28myb29 mutant completely lacking aliphatic glucosinolates and 1.8 higher on the single mutants with intermediate levels of aliphatic glucosinolates compared to wild type plants.
Identification of the Gene Encoding the 1,3-Mannosyltransferase (ALG3) in Arabidopsis and Characterization of Downstream N-Glycan Processing[W].
Henquet, M.G.L. ; Lehle, L. ; Schreuder, M.E.L. ; Rouwendal, G.J.A. ; Molthoff, J.W. ; Helsper, J.P.F.G. ; Krol, A.R. van der; Bosch, H.J. - \ 2008
The Plant Cell 20 (2008)6. - ISSN 1040-4651 - p. 1652 - 1664.
asparagine-linked oligosaccharides - deficient glycoprotein syndrome - unfolded protein response - yeast mutants deficient - endoplasmic-reticulum - saccharomyces-cerevisiae - congenital disorder - oligosaccharyltransferase complex - acetylglucosaminyltransferase
Glycosyltransferases are involved in the biosynthesis of lipid-linked N-glycans. Here, we identify and characterize a mannosyltransferase gene from Arabidopsis thaliana, which is the functional homolog of the ALG3 (Dol-P-Man: Man(5)GlcNAc(2)-PP-Dol alpha 1,3-mannosyl transferase) gene in yeast. The At ALG3 protein can complement a Delta alg3 yeast mutant and is localized to the endoplasmic reticulum in yeast and in plants. A homozygous T-DNA insertion mutant, alg3-2, was identified in Arabidopsis with residual levels of wild-type ALG3, derived from incidental splicing of the 11th intron carrying the T-DNAs. N- glycan analysis of alg3-2 and alg3-2 in the complex-glycan-less mutant background, which lacks N- acetylglucosaminyl-transferase I activity, reveals that when ALG3 activity is strongly reduced, almost all N-glycans transferred to proteins are aberrant, indicating that the Arabidopsis oligosaccharide transferase complex is remarkably substrate tolerant. In alg3-2 plants, the aberrant glycans on glycoproteins are recognized by endogenous mannosidase I and N- acetylglucosaminyltransferase I and efficiently processed into complex-type glycans. Although no high-mannose-type glycoproteins are detected in alg3-2 plants, these plants do not show a growth phenotype under normal growth conditions. However, the glycosylation abnormalities result in activation of marker genes diagnostic of the unfolded protein response.
RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis pathway, leads to parthenocarpic tomato fruits
Schijlen, E.G.W.M. ; Vos, C.H. de; Martens, S. ; Jonker, H.H. ; Rosin, F.M.A. ; Molthoff, J.W. ; Tikunov, Y.M. ; Angenent, G.C. ; Tunen, A.J. van; Bovy, A.G. - \ 2007
Plant Physiology 144 (2007)3. - ISSN 0032-0889 - p. 1520 - 1530.
transcription factor - male-sterility - arabidopsis-thaliana - male-fertility - flower color - tube growth - gene - pollen - petunia - expression
Parthenocarpy, the formation of seedless fruits in the absence of functional fertilization, is a desirable trait for several important crop plants, including tomato (Solanum lycopersicum). Seedless fruits can be of great value for consumers, the processing industry, and breeding companies. In this article, we propose a novel strategy to obtain parthenocarpic tomatoes by down-regulation of the flavonoid biosynthesis pathway using RNA interference (RNAi)-mediated suppression of chalcone synthase (CHS), the first gene in the flavonoid pathway. In CHS RNAi plants, total flavonoid levels, transcript levels of both Chs1 and Chs2, as well as CHS enzyme activity were reduced by up to a few percent of the corresponding wild-type values. Surprisingly, all strong Chs-silenced tomato lines developed parthenocarpic fruits. Although a relation between flavonoids and parthenocarpic fruit development has never been described, it is well known that flavonoids are essential for pollen development and pollen tube growth and, hence, play an essential role in plant reproduction. The observed parthenocarpic fruit development appeared to be pollination dependent, and Chs RNAi fruits displayed impaired pollen tube growth. Our results lead to novel insight in the mechanisms underlying parthenocarpic fruit development. The potential of this technology for applications in plant breeding and biotechnology will be discussed.
Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit
Schijlen, E.G.W.M. ; Vos, C.H. de; Jonker, H.H. ; Broeck, H.C. van den; Molthoff, J.W. ; Tunen, A.J. van; Martens, S. ; Bovy, A.G. - \ 2006
Plant Biotechnology Journal 4 (2006)4. - ISSN 1467-7644 - p. 433 - 444.
signal-transduction pathways - stilbene synthase gene - carcinoma cell-lines - heart-disease - biosynthetic-pathway - antioxidant activity - dietary flavonoids - petunia-hybrida - french paradox - male-sterility
Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards classes of potentially healthy flavonoids which are novel for tomato. Using structural flavonoid genes (encoding stilbene synthase, chalcone synthase, chalcone reductase, chalcone isomerase and flavone synthase) from different plant sources, we were able to produce transgenic tomatoes accumulating new phytochemicals. Biochemical analysis showed that the fruit peel contained high levels of stilbenes (resveratrol and piceid), deoxychalcones (butein and isoliquiritigenin), flavones (luteolin-7-glucoside and luteolin aglycon) and flavonols (quercetin glycosides and kaempferol glycosides). Using an online high-performance liquid chromatography (HPLC) antioxidant detection system, we demonstrated that, due to the presence of the novel flavonoids, the transgenic tomato fruits displayed altered antioxidant profiles. In addition, total antioxidant capacity of tomato fruit peel with high levels of flavones and flavonols increased more than threefold. These results on genetic engineering of flavonoids in tomato fruit demonstrate the possibilities to change the levels and composition of health-related polyphenols in a crop plant and provide more insight in the genetic and biochemical regulation of the flavonoid pathway within this worldwide important vegetable.
Lack of detrimental effects of Bacillus thuringiensis Cry toxins on the insect predator Chrysoperla carnea: a toxicological, histopathological, and biochemical analysis
Rodrigo-Simón, A. ; Maagd, R.A. de; Avilla, C. ; Bakker, P.L. ; Molthoff, J.W. ; González-Zamora, J. ; Ferré, J. - \ 2006
Applied and Environmental Microbiology 72 (2006)2. - ISSN 0099-2240 - p. 1595 - 1603.
border membrane-vesicles - helicoverpa-armigera lepidoptera - resistant transgenic plants - maize expressing cry1ab - crystal proteins - spodoptera-exigua - binding-sites - larval midgut - noctuidae - toxicity
The effect of Cry proteins of Bacillus thuringiensis on the green lacewing (Chrysoperla carnea) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed Helicoverpa armigera larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated H. armigera larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from Spodoptera exigua but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey
|Flavonoid pathway engineering for improved food quality
Schijlen, E.G.W.M. ; Vos, C.H. de; Jonker, H.H. ; Molthoff, J.W. ; Tunen, A.J. van; Bovy, A.G. - \ 2004
In: Book of abstracts of the 10th Netherlands Biotechnology Congress, Ede, The Netherlands, 11-12 March 2004. - - p. 88 - 88.
Activity of wild-type and hybrid Bacillus thuringiensis delta-endotoxins against Agrotis ipsilon
Maagd, R.A. de; Weemen-Hendriks, M. ; Molthoff, J.W. ; Naimov, S. - \ 2003
Archives of Microbiology 179 (2003). - ISSN 0302-8933 - p. 363 - 367.
insecticidal crystal proteins - cry1c domain-iii - spodoptera-exigua - binding - specificity - noctuidae - toxins - lepidoptera - resistance - site
Twelve Cry1 and two Cry9 ?-endotoxins fromBacillus thuringiensis were tested for their activity against black cutworm (Agrotis ipsilon).A. ipsilon was not susceptible to many toxins, but three toxins had significant activity. Cry9Ca was the most toxic, followed by Cry1Aa and Cry1Fb. Hybrids between these three active proteins were made by in vivo recombination and analyzed for activity againstA. ipsilon. Analysis of hybrids between Cry1Aa and Cry1Fb indicated that domain I of Cry1Aa protein was involved in its higher activity
Influence of Growth Conditions and Developmental Stage on N-Glycan Heterogeneity of Transgenic Immunoglobulin G and Endogenous Proteins in Tobacco Leaves
Elbers, I.J.W. ; Stoopen, G.M. ; Bakker, H. ; Stevens, L.H. ; Bardor, M. ; Molthoff, J.W. ; Jordi, W.J.R.M. ; Bosch, H.J. ; Lommen, A. - \ 2001
Plant Physiology 126 (2001). - ISSN 0032-0889 - p. 1314 - 1322.
rheumatoid-arthritis - leaf senescence - plant-cells - glycosylation - antibodies - igg - oligosaccharides - biosynthesis - glycoproteins - expression
Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant development and growth conditions on N-linked glycosylation. To investigate this, transgenic tobacco (Nicotiana tabacum cv Samsun NN) plants expressing a mouse immunoglobulin G antibody (MGR48) were grown in climate rooms under four different climate conditions, i.e. at 15°C and 25°C and at either low or high light conditions. N-glycans on plantibodies and soluble endogenous proteins were analyzed with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). Antibodies isolated from young leaves have a relatively high amount of high- mannose glycans compared with antibodies from older leaves, which contain more terminal N-acetylglucosamine. Senescence was shown to affect the glycosylation profile of endogenous proteins. The relative amount of N-glycans without terminal N-acetylglucosamine increased with leaf age. Major differences were observed between glycan structures on endogenous proteins versus those on antibodies, probably to be attributed to their subcellular localization. The relatively high percentage of antibody N-glycan lacking both xylose and fucose is interesting
Oral immunisation of naive and primed animals with transgenic potato tubers expressing LT-B
Lauterslager, T.G.M. ; Florack, D.E.A. ; Wal, T.J. van der; Molthoff, J.W. ; Langeveld, J.P.M. ; Bosch, D. ; Boersma, W.J.A. ; Hilgers, L.A.Th. - \ 2001
Vaccine 19 (2001). - ISSN 0264-410X - p. 2749 - 2755.
The efficacy of edible vaccines produced in potato tubers was examined in mice. Transgenic plants were developed by Agrobacterium tumefaciens-mediated transformation. The antigen selected was the non-toxic B subunit of the Escherichia coli enterotoxin (recLT-B). A synthetic gene coding for recLT-B was made and optimised for expression in potato tubers and accumulation in the endoplasmic reticulum. Introduction of this gene under control of the tuber-specific patatin promoter in potato plants resulted in the production of functional, i.e. Gm1-binding, recLT-B pentamers in tubers. Selected tubers containing about 13 g of recLT-B per gram fresh weight were used for immunisation. Subcutaneous immunisation with an extract of recLT-B tubers yielded high antibody titres in serum that were similar to those obtained with bacterial recLT-B. The efficacy of oral administration of recLT-B tubers was determined by measuring mucosal and systemic immune responses in naive and primed mice. Animals were primed by subcutaneous injection of an extract of recLT-B tuber plus adjuvant. Naive and primed mice were fed 5 g of tubers (~65 g of recLT-B) or were intubated intragastrically with 0.4 ml of tuber extract (~2 g of recLT-B). In naive mice, feeding recLT-B tubers or intubation of tuber extract did not induce detectable anti-LT antibody titres. In primed animals, however, oral immunisation resulted in significant anti-LT IgA antibody responses in serum and faeces. Intragastric intubation of tuber extract revealed higher responses than feeding of tubers. These results indicate clearly that functional recLT-B can be produced in potato tubers, that this recombinant protein is immunogenic and that oral administration thereof elicits both systemic and local IgA responses in parentally primed, but not naive, animals.
Galactose-extended glycans of antibodies produced by transgenic plants
Bakker, H. ; Bardor, M. ; Molthoff, J.W. ; Gomord, V. ; Elbers, I. ; Stevens, L.H. ; Jordi, W. ; Lommen, A. ; Faye, L. ; Lerouge, P. ; Bosch, D. - \ 2001
Proceedings of the National Academy of Sciences of the United States of America 98 (2001)5. - ISSN 0027-8424 - p. 2899 - 2904.
Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that β1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human β1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal β1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal β1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human β1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.
Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco
Stevens, L.H. ; Stoopen, G.M. ; Elbers, I.J.W. ; Molthoff, J.W. ; Bakker, H.A.C. ; Lommen, A. ; Bosch, D. ; Jordi, W. - \ 2000
Plant Physiology 124 (2000)1. - ISSN 0032-0889 - p. 173 - 182.
Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant physiology and plant development on the yield and quality of the heterologous proteins produced in plants. To investigate this, tobacco (Nicotiana tabacum cv Samsun NN) was transformed with a single construct that contained behind constitutive promotors the light- and heavy-chain genes of a mouse antibody. The in planta stability of the antibody was analyzed in transgenic plants that were grown under high and low irradiation at 15°C and 25°C. High-light conditions favored the production of biomass, of total soluble protein, and of antibody. The plants grown at 25°C developed faster and contained less antibody per amount of leaf tissue than the plants grown at 15°C. Both endogenous protein and antibody content showed a strong decline during leaf development. The heavy chains of the antibody underwent in planta degradation via relatively stable fragments. In vitro incubations of purified plantibody with leaf extracts of wild-type tobacco indicated the involvement of acidic proteases. It is interesting that the same antibody produced by mouse hybridoma cells exhibited higher stability in this in vitro assay. This may be explained by the assumption that the plant type of N-glycosylation contributes less to the stability of the antibody than the mouse-type of N-glycosylation. The results of this study indicate that proteolytic degradation during plant development can be an important factor affecting yield and homogeneity of heterologous protein produced by transgenic plants.
|'Plantibodies': flexible genes for engineering resistance.
Schots, A. ; Roosien, J. ; Schouten, A. ; Wilmink, A. ; Engelen, F.A. van; Molthoff, J. ; Borst-Vrenssen, T. ; Jong, I. de; Pomp, R. ; Overmars, H. ; Boer, J. de; Bosch, D. ; Stiekema, W. ; Gommers, F.J. ; Bakker, J. - \ 1996
In: Abstract 3rd Int. Nematology Congr., Guadeloupe - p. 70 - 70.
|(Pl)antibodies; a possible alternative in the absence of natural host-plant resistance genes.
Schouten, A. ; Roosien, J. ; Boer, J.M. de; Engelen, F.A. van; Wilmink, A. ; Jong, G.A.M. de; Borst-Vrenssen, A.W.M. ; Pomp, H. ; Overmars, H.A. ; Molthoff, J.W. ; Bosch, D.H. ; Gommers, F.J. ; Stiekema, W.J. ; Schots, A. ; Bakker, J. - \ 1996
In: Abstract 13th Triennial Conf. European Association for Potato Research, Veldhoven - p. 701 - 701.