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  • 1
    Electronic Resource
    Electronic Resource
    Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants (1991)
    Springer
    Plant molecular biology 17 (1991), S. 691-699 
    Details
    ISSN: 1573-5028
    Keywords: chimaeric gene ; granule-bound starch synthase ; sugar inducibility ; Solanum tuberosum L. ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5′ upstream sequence of the granule-bound starch synthase gene from potato and the β-glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00037054
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  • 2
    Electronic Resource
    Electronic Resource
    Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth (1994)
    Springer
    Plant molecular biology 26 (1994), S. 1759-1773 
    Details
    ISSN: 1573-5028
    Keywords: antisense RNA ; field trial ; granule-bound starch synthase ; Solanum tuberosum L. ; transgenic plants ; tuber development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00019490
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  • 3
    Electronic Resource
    Electronic Resource
    GBSS T-DNA inserts giving partial complementation of the amylose-free potato mutant can also cause co-suppression of the endogenous GBSS gene in a wild-type background (1996)
    Springer
    Plant molecular biology 31 (1996), S. 731-739 
    Details
    ISSN: 1573-5028
    Keywords: complementation ; expression ; co-suppression ; granule-bound starch synthase ; Solanum tubersoum L. ; transgenic inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The wild-type gene encoding granule-bound starch synthase (GBSS) is capable of both complementing the amylosefree (amf) potato mutant and inhibiting the endogenous GBSS gene expression in wild-type potato. Co-suppression of the endogenous GBSS gene, easily visualised by staining the starch with iodine, occurred when the full-size GBSS sequence (genomic), GBSS cDNA or even the mutant amf allele were introduced into the wild-type potato. Conversely, introduction of the GBSS promoter sequence alone, did not result in co-suppression in the 80 analysed transformants. Neither the orientation of the GBSS gene with respect to kanamycin resistance nor the presence of an enhancer influenced the frequency of plants showing a co-suppression phenotype. After crossing a partially complemented amf mutant with a homozygous wild-type plant, the F1 offspring segregated into plant phenotypes with normal and decreased expression of the GBSS gene. This decreased expression correlated with the presence of a linked block of five T-DNA inserts which was previously shown to be correlated with partial complementation of the amf mutant. This crossing experiment indicates that co-suppression can cause inhibition of gene expression of both inserted and endogenous wild-type GBSS genes. The frequency of partially complemented amf plants was equal to the frequency of co-suppressed wild types when a construct, with an enhancer in front of the GBSS promoter, was used (pWAM 101E). This might suggest that partial complementation of the amf genotype caused by unstable expression of the transgene can be overcome by inserting an enhancer in front of the GBSS promoter.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00019461
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  • 4
    Electronic Resource
    Electronic Resource
    Developmental changes of enzymes involved in conversion of sucrose to hexose-phosphate during early tuberisation of potato (1997)
    Springer
    Planta 202 (1997), S. 220-226 
    Details
    ISSN: 1432-2048
    Keywords: Key words: Fructokinase ; Hexokinase ; Invertase ; Solanum ; Sucrose synthase ; Tuberisation (sucrose metabolism)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. A highly synchronised in-vitro tuberisation system, based on single-node cuttings containing an axillary bud, was used to investigate the activity patterns of enzymes involved in the conversion of sucrose to hexose-phosphates during stolon-to-tuber transition of potato (Solanum tuberosum L.). Two different non-tuberising systems were included to distinguish between changes that are or are not tuber-specific. At tuberisation the activity of soluble acid invertase decreased (13-fold) and of sucrose synthase increased (12-fold). The activity of both enzymes remained unchanged in the non-tuberising treatments. Based on the opposite patterns and large difference in activity of these two sucrolytic enzymes, we conclude that sucrose synthase constitutes the predominant route of sucrose breakdown after tuber initiation. During the period before tuberisation, the activity of cell-wall-bound invertase and of hexokinase showed a highly positive correlation (r 2 = 0.96 in all the three treatments, suggesting coordinated coarse control of both enzyme activities. After the onset of tuberisation cell-wall-bound invertase activity decreased to a very low level, a change not observed in the non-tuberising systems, indicating that cell-wall-bound invertase is presumably not involved in the unloading mechanism and/or short-distance transport of sucrose within the perimedulla of growing tubers. The overall activity of fructokinase and of hexokinase both showed a fourfold increase after tuber initiation, but remained unchanged in the non-tuberising systems. The increase of fructokinase suggests that the phosphorylation of fructose by fructokinase down-regulates the cytosolic fructose content in order to maintain a high sucrose-synthase-catalysed net flux of sucrose to phosphorylated hexoses during rapid tuber growth. The increase of total glucose-phosphorylating potential could be a response to the tuberisation-related starch accumulation process. The activity of UDP-glucose pyrophosphorylase showed no developmental change. The level of UDP-glucose pyrophosphorylase activity is very likely the result of metabolic regulation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004250050122
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  • 5
    Electronic Resource
    Electronic Resource
    Physiological and genetic control of tuber formation (1999)
    Springer
    Potato research 42 (1999), S. 313-331 
    Details
    ISSN: 1871-4528
    Keywords: tuberization ; stolon formation ; hormones ; morphological changes ; gibberellins ; QTL mapping ; fingerpringting ; carbohydrate metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Tuber formation is a plalstic and complex, but well-orchestrated sequences of morphological. physiological and biochemical events. The physiological control mechanisms of this sequence of events may involve many hormones, but certainly gibberellic acids play a dominant role: they affecft most steps and are influenced by inducing or non-inducing conditions in a manner consistent with effects of these conditions on tuber induction. The genetic control is also complex. Numerous cDNA fragments have been isolated which are specifically expressed during tuberization. They can be used in anti-sense orientation in transgenic plants to thest their possible role in tuberization. In addition these cDNA fragments are used as genetic marker loci in QTL mapping studies and serve as candidate genes to exlain phenotypic variation. Profiles of the importance of QTLs over time may be combined with expression profiles of candidate genes. Such novel approaches offer unique opportunities for synergism between physiology, molecular biology and genetics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02357860
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  • 6
    Electronic Resource
    Electronic Resource
    Cloning and expression analysis of a potato cDNA that encodes branching enzyme evidence for co-expression of starch biosynthetic genes (1991)
    Springer
    Molecular genetics and genomics 230 (1991), S. 39-44 
    Details
    ISSN: 1617-4623
    Keywords: Branching enzyme ; Potato ; Starch synthesis ; Sucrose induction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary One of the key enzymes involved in the formation of amylopectin, which is the major component of starch, is branching enzyme. A cDNA for potato branching enzyme was cloned by screening a tuber-specific cDNA expression library using an antiserum directed against a denatured preparation of the protein. Complementation of an Escherichia coli strain deficient in branching enzyme was achieved using a construct derived from this clone. Analysis of the expression of the gene in potato revealed a close association with conditions favouring starch biosynthesis. The expression pattern of the gene coding for potato branching enzyme, as analyzed at the mRNA level, closely resembles that of 4GPase S, a gene coding for one of the subunits of ADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. This raises the possibility that enzymes involved in the pathway are coordinately regulated at the transcriptional level.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00290648
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  • 7
    Electronic Resource
    Electronic Resource
    Factors affecting the inhibition by antisense RNA of granule-bound starch synthase gene expression in potato (1995)
    Springer
    Molecular genetics and genomics 246 (1995), S. 745-755 
    Details
    ISSN: 1617-4623
    Keywords: Antisense effect ; Granule-bound starch synthase (GBSS) ; Introns Promoter ; T-DNA copy number
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Inhibition of expression of specific genes by means of antisense RNA is widely used, although little information is available regarding conditions that affect the efficacy of inhibition. In this study, inhibition of granule-bound starch synthase (GBSS), a key enzyme in starch biosynthesis, is used as a model system. Eleven antisense constructs derived from the full-length GBSS cDNA, the genomic GBSS coding region (gDNA) or fragments of each of these sequences, were analysed with respect to their inhibitory effect. Introduction of full-length gDNA constructs yielded a lower percentage of transgenic clones showing complete inhibition than did introduction of the full-length cDNA constructs. This may be caused by a lower antisense binding capacity of the former due to the relatively low GC content in intron sequences present in the gDNA constructs. The presence of multiple T-DNA insertions was related to a higher degree of inhibition. Putative polyadenylation signals on the antisense strand of the GBSS gene resulted in a premature stop of transcription of some of the antisense genes, as demonstrated by the expression of smaller antisense RNA transcripts. Introduction of antisense constructs driven by the promoter of the (target) GBSS gene resulted in a higher percentage of clones with complete inhibition than introduction of antisense constructs driven by the 35S CaMV promoter. Complete antisense inhibition was achieved in 25% of the clones carrying the antisense construct pKGBA 50, which is based on the GBSS promoter and the full-length GBSS cDNA. Thus, it is concluded that the use of pKGBA50 is very suitable for the modification of the composition of potato tuber starch via antisense RNA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00290722
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  • 8
    Electronic Resource
    Electronic Resource
    Sequence of the structural gene for granule-bound starch synthase of potato (Solarium tuberosum L.) and evidence for a single point deletion in the amf allele (1991)
    Springer
    Molecular genetics and genomics 228 (1991), S. 240-248 
    Details
    ISSN: 1617-4623
    Keywords: Frameshift ; Potato ; Starch ; Transit peptide ; Waxy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The genomic sequence of the potato gene for starch granule-bound starch synthase (GBSS; “waxy protein”) has been determined for the wild-type allele of a monoploid genotype from which an amylose-free (amf) mutant was derived, and for the mutant part of the amf allele. Comparison of the wild-type sequence with a cDNA sequence from the literature and a newly isolated cDNA revealed the presence of 13 introns, the first of which is located in the untranslated leader. The promoter contains a G-box-like sequence. The deduced amino acid sequence of the precursor of GBSS shows a high degree of identity with monocot waxy protein sequences in the region corresponding to the mature form of the enzyme. The transit peptide of 77 amino acids, required for routing of the precursor to the plastids, shows much less identity with the transit peptides of the other waxy preproteins, but resembles the hydropathic distributions of these peptides. Alignment of the amino acid sequences of the four mature starch synthases with the Escherichia coli glgA gene product revealed the presence of at least three conserved boxes; there is no homology with previously proposed starch binding domains of other enzymes involved in starch metabolism. We report the use of chimeric constructs with wild-type and amf sequences to localize, via complementation experiments, the region of the amf allele in which the mutation resides. Direct sequencing of polymerase chain reaction products confirmed that the amf mutation is a deletion of a single AT basepair in the region coding for the transit peptide. Premature termination of translation as a result of this frameshift mutation results in a small peptide. However, a protein reacting with anti-GBSS serum, slightly larger than the wild-type mature GBSS, can be detected in a membrane fraction from amylose-free tubers. A possible explanation for this phenomenon will be discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00282472
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  • 9
    Electronic Resource
    Electronic Resource
    The protein composition of the cytoplasmic membrane of aerobically and anaerobically grownEscherichia coli (1984)
    Springer
    Journal of bioenergetics and biomembranes 16 (1984), S. 295-307 
    Details
    ISSN: 1573-6881
    Keywords: Escherichia coli ; crossed immunoelectrophoresis ; cytoplasmic membrane proteins ; aerobic growth ; anaerobic growth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The protein composition of the cytoplasmic membranes ofEscherichia coli, grown aerobically and anaerobically on a glucose minimal medium at pH 7.0, were analyzed by crossed immunoelectrophoresis. Qualitative differences are limited to only two proteins: nitrate reductase (E.C. 1.7.99.4) is absent under aerobic growth conditions, whereas an unidentified protein, with a molecular weight of 81,500 and located at the inner side of the cytoplasmic membrane, is synthesized only in the presence of oxygen. Quantitative differences are observed for many proteins: the ratio of the amount of a specific protein present in cells grown anaerobically and aerobically was, for four proteins, between 0.3 and 1; for 25 proteins, between 1 and 3; and for five proteins, larger than 5.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00744282
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  • 10
    Electronic Resource
    Electronic Resource
    Expression of wild-type GBSS transgenes in the off-spring of partially and fully complementedamylose-free transformants of potato (1996)
    Springer
    Molecular breeding 2 (1996), S. 211-218 
    Details
    ISSN: 1572-9788
    Keywords: complementation ; granule-bound starch synthase ; Solanum tuberosum L. ; expression ; transgenic inheritance ; ploidy level
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Theamylose-free (amf) potato mutant can easily be complemented through introduction of the wild-type gene coding for granule-bound starch synthase (GBSS). After iodine staining the starch of theamf mutant is red whereas that of the wild type and the complementedamf mutant is blue. The level of complementation of selected transformants and their sexual off-spring after backcrossing withamf was investigated using sporophytic tuber cells and gametophytic microspore cells. Two diploid and two tetraploid transformants with full complementation demonstrated the expected segregation patterns of 1:1 (one active insert) or 3:1 (two independently segregating active inserts) in the microspores and in the F1 offspring based on staining of tubers. All expected genotypes in the F1 generation were found, based on microspore segregation patterns of the individual F1 plants. Two transformants with partial complementation (mixed phenotypes) were investigated. One of them, B1, was tetraploid and duplex for the GBSS insert, which had originated through mitotic doubling of the transformed diploid cells. In the F1 generation three phenotypic classes were found:amf, fully complemented and partially complemented. The latter two classes exist independently of a simplex or duplex gene status. The second transformant with partial complementation, B10, appeared to have a complex molecular composition. One cluster of five transgenes caused the partial complementation. Fully and partially complemented phenotypic classes were found after crossing B10 with theamf mutant. Indications were found that the ploidy level of the tissue in which the genes were introduced and expressed played an important role. Firstly, partial complementation was found after transformation of the diploid and not of the tetraploidamf genotypes. Secondly, the level of complementation was higher in tissue with lower ploidy levels, as illustrated by the colour of the starch inin vitro tubers (2x–4x cells) versus field-grown tubers (16x–64x).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00564198
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