Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants

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 -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.     

Evidence that amylose synthesis occurs within the matrix of the starch granule in potato tubers

Transgenic potatoes expressing reduced levels of granule-bound starch synthase I (GBSSI) have been used to investigate whether the synthesis of amylose occurs at the surface of the starch granule or within the matrix formed by the synthesis and organization of amylopectin. Amylose in these potatoes is wholly or largely confined to a central region of the granule. Consequently this core region stains blue with iodine whereas the peripheral zone stains red. By making extensive measurements of the relative sizes of the granules and their blue-staining cores in tubers over a range of stages of development, we have established that the blue core increases in size as the granule grows. The extent of the increase in size of the blue core is greater in potatoes with higher levels of GBSSI. These data show that amylose synthesis occurs within the matrix of the granule, and are consistent with the idea that the space available in the matrix may be an important determinant of the amylose content of storage starches.     

Composition of endogenous alleles can influence the level of antisense inhibition of granule-bound starch synthase gene expression in tetraploid potato plants

The T-DNA composition was analysed of twelve potato genotypes obtained after transforming a tetraploid cultivar with an antisense granule-bound starch synthase (GBSSI) gene. In five transformants (labelled TB50 nos.) the antisense GBSSI gene was driven by the CaMV 35S promoter, while in the remaining seven (labelled TBK50 nos.) the GBSSI promoter was used. In these twelve transformants the antisense effect on amylose production in potato tuber starch ranged from complete suppression to no discernible inhibition, and the number of T-DNA insertions ranged from one to at least fifteen. The antisense effect of individual T-DNA loci in progeny of these transformants was studied. Progeny containing a single T-DNA showed no inhibition of GBSSI activity. Only multiple, linked T-DNA insertions resulted in substantial antisense inhibition. T-DNA fragments present in duplex in selfed progeny resulted in a larger antisense effect than that in the parent (which contained the T-DNA insertions in simplex). Furthermore, the antisense effects of some T-DNA-containing linkage groups were influenced by the composition of endogenous GBSSI alleles. For practical breeding this implies that (1) the efficiency of obtaining primary potato transformants showing complete inhibition of GBSSI gene expression by antisense RNA is genotype-dependent, and (2) many transformants have to be produced per genotype to be able to select plants with maximum suppression of GBSSI and a minimum number of T-DNA loci.     

Potato granule-bound starch synthase promoter-controlled GUS expression: regulation of expression after transient and stable transformation

Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the -glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosumSolanum brevidens, Nicotiana tabacum andArabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.     

Complementation of the amylose-free starch mutant of potato (Solanum tuberosum.) by the gene encoding granule-bound starch synthase

Summary Agrobacterium rhizogenes-mediated introduction of the wild-type allele of the gene encoding granulebound starch synthase (GBSS) into the amylose-free starch mutantamf of potato leads to restoration of GBSS activity and amylose synthesis, which demonstrates thatAmf is the structural gene for GBSS. Amylose was found in columella cells of root tips, in stomatal guard cells, tubers, and pollen, while in the control experiments using only vector DNA, these tissues remained amylose free. This confirms the fact that, in potato, GBSS is the only enzyme responsible for the presence of amylose, accumulating in all starch-containing tissues. Amylose-containing transformants showed no positive correlation between GBSS activity and amylose content, which confirms that the former is not the sole regulating factor in amylose metabolism.     

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

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.     

Progress in understanding the biosynthesis of amylose

The storage of glucose in insoluble granules is a distinctive feature of plant cells. Biosynthesis of amylose, the minor low molecular mass fraction of starch occurs from ADP-glucose. This takes place within the polysaccharide matrix through the action of granule-bound starch synthase, the major protein associated with the granule. Recently, amylose has been successfully synthesized in vitro from purified granules. Two models have been proposed to explain the mechanism of amylose synthesis in plants. The first calls for priming of synthesis through small-size malto-oligosaccharides. The second suggests that glucans are extended by granule-bound starch synthase from a high molecular mass primer present within the granule. This extension is terminated through cleavage to produce amylose. This process is subsequently repeated to give several rounds of amylose synthesis.     

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

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.     

Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs

Summary Granule-bound starch synthase [GBSS; EC 24.1.21] determines the presence of amylose in reserve starches. Potato plants were transformed to produce antisense RNA from a gene construct containing a full-length granule-bound starch synthase cDNA in reverse orientation, fused between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The construct was integrated into the potato genome by Agrobacterium rhizogenes-mediated transformation. Inhibition of GBSS activity in potato tuber starch was found to vary from 70% to 100%. In those cases where total suppression of GBSS activity was found both GBSS protein and amylose were absent, giving rise to tubers containing amylose-free starch. The variable response of the transformed plants indicates that position effects on the integrated sequences might be important. The results clearly demonstrate that in tubers of potato plants which constitutively synthesize antisense RNA the starch composition is altered.     

Expression of wild-type GBSS transgenes in the off-spring of partially and fully complementedamylose-free transformants of potato

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).     

Variation in the primary structure ofwaxy proteins (granule-bound starch synthase) in diploid cereals

The molecular weights ofwaxy proteins, by SDS-PAGE, and the N-terminal amino acid sequences of mature protein and of V8 protease-induced fragments were determined in diploid cereals. The homology of the primary structure was relatively high among cereals examined here, and there appeared to be a common sequence, V-F-V-G-A-E-M-A, in the vicinity of the N terminus. Based on the amino acid sequences, these cereals could be divided into two groups, including corn and rice in one and diploid wheat, fourAegilops species, rye, and barley in the other. In diploid wheat andAegilops species there were substitutions of amino acids in the primary structure. Variations of this sort suggest that the primary structure ofwaxy proteins would provide clues to the phylogenetic relations in the wheat group.     

Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.)

Summary An amylose-free potato mutant was isolated after screening 12,000 minitubers. These minitubers had been induced on stem segments of adventitious shoots, which had been regenerated on leaf explants of a monoploid potato clone after Röntgen-irradiation. The mutant character is also expressed in subterranean tubers and in microspores. Starch granules from the mutant showed a strongly reduced activity of the granule bound starch synthase and loss of the major 60 kd protein from the starch granules.     

Sucrose-inducible expression of hepatitis B surface antigen using potato granule-bound starch synthase promoter

NT-1 cells of tobacco (Nicotiana tabacum L.) were transformed with pGBSSHBS and pGBSSHER expression cassettes wherein expression of hepatitis B surface antigen (HBsAg) was driven by potato granule-bound starch synthase (GBSS) promoter. The transformed nature of the cells was confirmed by PCR analysis. Expression of HBsAg was confirmed by RT-PCR and Western blotting and levels of expression were assayed by ELISA. Transformed cell lines exhibited a sucrose-inducible pattern of HBsAg expression. NT-1 medium supplemented with 175 mmol L⁻¹ sucrose gave the highest HBsAg expression of 198 ng g⁻¹ FW after 8 days of induction. Different sugars, for example glucose, fructose, and palatinose, were also tested to study the inducible nature of GBSS promoter. The results demonstrate that potato GBSS promoter can be used in heterologous host systems like tobacco NT-1 cell suspension cultures for sucrose-inducible expression of recombinant proteins.     

Mutation loci and intragenic selection marker of the granule-bound starch synthase gene in waxy maize

Four pairs of specific PCR primers have been designed on the basis of the sequence of the granule-bound starch synthase gene (GBSS; dominant non-waxy gene Wx) and used to amplify its homologous sequence from thirteen waxy and two non-waxy inbred lines. Results from electrophoresis indicated that the recessive waxy gene was wx, derived from the dominant non-waxy gene Wx by mutation at its 3′ end. The sequence of the mutated 3′ end was amplified by the TAIL-PCR technique. Sequence alignment showed that the mutation of the wx gene was caused by transposition of the aldehyde dehydrogenase gene rf2. Two pairs of specific primers were designed on the basis of the sequence difference between the dominant gene Wx and its mutated recessive allele wx and used as intragenic selection markers to identify individual plants of genotypes WxWx, Wxwx, and wxwx by PCR amplification from the segregating population of the F₂ generation crossed between waxy and non-waxy inbred lines. Iodine solution staining and starch component assay showed that all the 35 F₂ plants identified as genotype WxWx produced non-waxy kernels of the F₃ generation and that all 33 F₂ plants identified as genotype wxwx produced waxy kernels of the F₃ generation. This result can be used to improve the selection efficiency of waxy maize breeding and for selection of other single genes and major polygenes.     

Expression of a wild-type GBSS gene introduced into an amylose-free potato mutant by Agrobacterium tumefaciens and the inheritance of the inserts at the microsporic level

Granule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Transformation of a diploid amylose-free (amf) potato mutant with the gene encoding GBSS leads to the restoration of amylose synthesis. Transformants were obtained which had wild-type levels of both GBSS activity and amylose content. It proved to be difficult to increase the amylose content above that of the wild-type potato by the introduction of additional copies of the wild-type GBSS gene. Staining of starch with iodine was suitable for investigating the degree of expression of the inserted GBSS gene in transgenic amf plants. Of the 19 investigated transformants, four had only red-staining starch in tubers indicating that no complementation of the amf mutation had occured. Fifteen complemented transformants had only blue-staining starch in tubers or tubers of different staining categories (blue, mixed and red), caused either by full or partial expression of the inserted gene. Complementation was also found in the microspores. The segregation of blue- and red-staining microspores was used to analyse the inheritance of the introduced GBSS genes. A comparison of the results from microspore staining and Southern hybridisation indicated that, in three tetraploid transgenics, the gene was probably inserted before (duplex), and in all others after, chromosome doubling (simplex). The partial complementation was not due to methylation of the HPAII/MSPI site in the promoter region. Partially complemented plants had low levels of mRNA as was found when the GBSS expression levels were inhibited by anti-sense technology.