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.     

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).     

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.     

Expression of a cassava granule-bound starch synthase gene in the amylose-free potato only partially restores amylose content

Granule-bound starch synthase I (GBSS I) is responsible for the synthesis of amylose in starch granules. A heterologous cassava GBSS I gene was tested for its ability to restore amylose synthesis in amylose-free (amf) potato mutants. For this purpose, the cassava GBSS I was equipped with different transit peptides. In addition, a hybrid containing the potato transit peptide, the N-terminal 89 amino acids of the mature potato GBSS I, and the C-terminal part of cassava GBSS I was prepared. The transgenic starches were first analysed by iodine staining. Only with the hybrid could full phenotypic complementation of the amf mutation be achieved in 13% of the plants. Most transformants showed partial complementation, but interestingly the size of the blue core was similar in all granules derived from one tuber of a given plant. The amylose content was only partially restored, up to 60% of wild-type values or potato GBSS I-complemented plants; however, the GBSS activity in these granules was similar to that found in wild-type ones. From this, and the observation that the hybrid protein (a partial potato GBSS I look-alike) performs best, it was concluded that potato and cassava GBSS I have different intrinsic properties and that the cassava enzyme is not fully adapted to the potato situation.     

Formation and Deposition of Amylose in the Potato Tuber Starch Granule Are Affected by the Reduction of Granule-Bound Starch Synthase Gene Expression

The synthesis of amylose in amyloplasts is catalyzed by granule-bound starch synthase (GBSS). GBSS gene expression was inhibited via antisense RNA in Agrobacterium rhizogenes-transformed potato plants. Analysis of starch production and starch granule composition in transgenic tubers revealed that reduction of GBSS activity always resulted in a reduction of the production of amylose. Field experiments, performed over a 2-year period, showed that stable inhibition of GBSS gene expression can be obtained. Microscopic evaluation of iodine-stained starch granules was shown to be a sensitive system for qualitative and quantitative examination of amylose formation in starch granules of transgenic potato tubers. In plants showing inhibition of GBSS gene expression, the reduced amylose content in tuber starch was not a consequence of a lower amylose content throughout the entire starch granule. Starch granules of transgenic tubers were found to contain amylose at a percentage similar to wild-type starch in a core of varying size at the hilum of each granule. This indicated that reduced GBSS gene expression results in amylose formation in a restricted zone of the granules. The size of this zone is suggested to be dependent on the GBSS protein level. During development of the granules, the available GBSS protein is thought to become limiting, resulting in the formation of starch that lacks amylose. RNA gel blot analysis of tuber tissue showed that inhibition of GBSS gene expression resulted in a reduced GBSS mRNA level but did not affect the expression level of other starch synthesizing enzymes. Antisense RNA could only be detected in leaf tissue of the transgenic plants.     

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.     

Sequence of the structural gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele

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.     

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.     

Accumulation of multiple-repeat starch-binding domains (SBD2–SBD5) does not reduce amylose content of potato starch granules

This study investigates whether it is possible to produce an amylose-free potato starch by displacing the amylose enzyme, granule-bound starch synthase I (GBSSI), from the starch granule by engineered, high-affinity, multiple-repeat family 20 starch-binding domains (SBD2, SBD3, SBD4, and SBD5). The constructs were introduced in the amylose-containing potato cultivar (cv. Kardal), and the starches of the resulting transformants were compared with those of SBD2-expressing amylose-free (amf) potato clones. It is shown that a correctly sized protein accumulated in the starch granules of the various transformants. The amount of SBD accumulated in starch increased progressively from SBD to SBD3; however, it seemed as if less SBD4 and SBD5 was accumulated. A reduction in amylose content was not achieved in any of the transformants. However, it is shown that SBDn expression can affect physical processes underlying granule assembly, in both genetic potato backgrounds, without altering the primary structure of the constituent starch polymers and the granule melting temperature. Granule size distribution of the starches obtained from transgenic Kardal plants were similar to those from untransformed controls, irrespective of the amount of SBDn accumulated. In the amf background, granule size is severely affected. In both the Kardal and amf background, apparently normal oval-shaped starch granules were composed of multiple smaller ones, as evidenced from the many “Maltese crosses” within these granules. The results are discussed in terms of different binding modes of SBD.     

Isolation and characterization of potato-tomato somatic hybrids using an amylose-free potato mutant as parental genotype

Summary Using different genotypes of tomato and diploid potato, possessing alien selectable markers as well as endogenous markers, very high frequencies of protoplast fusion hybrids were obtained. One endogenous genetic marker, the amylose-free (amf) mutant of potato, was helpful not only for the confirmation of fusion products but also for the study of genetic complementation and the segregation of amylose-free starch in microspores. Cytological analysis of the fusion hybrids indicated that except for one which was hexaploid, all of them had a perfectly balanced chromosome number of allotetraploid constitution (2n = 4x = 48). Despite normal chromosome pairing and a diploid behaviour, the microspores in some of the fusion hybrids segregated for the recessive amf-locus. This anomalous segregation of a recessive character in these hybrids was shown not to be due to chromosome elimination or to the absence of the wild-type tomato Amf gene. Although all fusion hybrids were totally sterile, the hexaploid produced stainable pollen and berries with badly developed seeds. Embryo rescue has so far failed to produce backcross progeny.     

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.     

Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch

One isoform of the branching enzyme (BE; EC 2.4.1.18) of potato (Solarium tuberosum L.) is known and catalyses the formation of α-1,6 bonds in a glucan chain, resulting in the branched starch component amylopectin. Constructs containing the antisense or sense-orientated distal 1.5-kb part of a cDNA for potato BE were used to transform the amylose-free (amf) mutant of potato, the starch of which stains red with iodine. The expression of the endogenous BE gene was inhibited either largely or fully as judged by the decrease or absence of the BE mRNA and protein. This resulted in a low percentage of starch granules with a small blue core and large red outer layer. There was no effect on the amylose content, degree of branching or λ_max of the iodine-stained starch. However, when the physico-chemical properties of the different starch suspensions were assessed, differences were observed, which although small indicated that starch in the transformants was different from that of theamf mutant.     

Creation of a potato mutant lacking the starch branching enzyme gene StSBE3 that was generated by genome editing using the CRISPR/dMac3-Cas9 system

The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.     

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.     

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.     

Microbial starch-binding domains as a tool for targeting proteins to granules during starch biosynthesis

Modification of starch biosynthesis pathways holds an enormous potential for tailoring granules or polymers with new functionalities. In this study, we explored the possibility of engineering artificial granule-bound proteins, which can be incorporated in the granule during biosynthesis. The starch-binding domain (SBD)-encoding region of cyclodextrin glycosyltransferase from Bacillus circulans was fused to the sequence encoding the transit peptide (amyloplast entry) of potato granule-bound starch synthase I (GBSS I). The synthetic gene was expressed in the tubers of two potato cultivars (cv. Kardal and cv. Karnico) and one amylose-free (amf) potato mutant. SBDs accumulated inside starch granules, not at the granule surface. Amylose-free granules contained 8 times more SBD (estimated at ca. 1.6% of dry weight) than the amylose-containing ones. No consistent differences in physicochemical properties between transgenic SBD starches and their corresponding controls were found, suggesting that SBD can be used as an anchor for effector proteins without having side-effects. To test this, a construct harbouring the GBSS I transit peptide, the luciferase reporter gene, a PT-linker, and the SBD (in frame), and a similar construct without the linker and the SBD, were introduced in cv. Kardal. The fusion protein accumulated in starch granules (with retainment of luciferase activity), whereas the luciferase alone did not. Our results demonstrate that SBD technology can be developed into a true platform technology, in which SBDs can be fused to a large choice of effector proteins to generate potato starches with new or improved functionalities.     

The dosage effect of the wildtype GBSS allele is linear for GBSS activity but not for amylose content: absence of amylose has a distinct influence on the physico-chemical properties of starch

A gene-dosage population was obtained by crossing two genotypes that were duplex for the GBSS allele. Nulliplex, simplex, duplex or triplex/quadruplex plants could be identified by monitoring the segregation of red and blue microspores after staining with iodine. GBSS activity was significantly different for all groups and showed an almost linear dosage effect for the wildtype GBSS gene. A dosage effect was found for amylose content that was not linear. The amylose content was similar for both the duplex and triplex/quadruplex group. Within the simplex group, differences in amylose content were found, which might be due to a different genetic background. There was no linear correlation between GBSS activity and amylose content. A certain level of GBSS activity led to a maximum amount of amylose, and further increase in GBSS activity did not result in a further increase in amylose content. The presence of one or more wildtype GBSS allele(s), and therefore the presence of amylose in the starch granules, had a great influence on the physico-chemical properties of the starch suspensions.     

Tracking sulfur and phosphorus within single starch granules using synchrotron X-ray microfluorescence mapping

Background

Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2–0.5%) and proteins (0.1–0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis.

Methods

Synchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme.

Results

Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3–5 μm in diameter.

Conclusions

Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before.

General significance

μXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds.