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.     

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.     

Mutan produced in potato amyloplasts adheres to starch granules

Production of water-insoluble mutan polymers in Kardal potato tubers was investigated after expression of a full-length (GtfI) and a truncated mutansucrase gene referred to as GtfICAT (GtfI without glucan-binding domain) from Streptococcus downei. Subsequent effects on starch biosynthesis at the molecular and biochemical levels were studied. Expression of the GtfICAT gene resulted in the adhesion of mutan material on starch granules, which stained red with erythrosine, and which was hydrolysed by exo-mutanase. In addition, GtfICAT-expressing plants exhibited a severely altered tuber phenotype and starch granule morphology in comparison to those expressing the full-length GtfI gene. In spite of that, no structural changes at the starch level were observed. Expression levels of the sucrose-regulated, AGPase and GBSSI genes were down-regulated in only the GTFICAT transformants, showing that GtfICAT expression interfered with the starch biosynthetic pathway. In accordance with the down-regulated AGPase gene, a lower starch content was observed in the GTFICAT transformants. Finally, the rheological properties of the GTFICAT starches were modified; they showed a higher retrogradation during cooling of the starch paste.     

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.     

Expression of an amylosucrase gene in potato results in larger starch granules with novel properties

Main conclusion

Expression of amylosucrase in potato resulted in larger starch granules with rough surfaces and novel physico-chemical properties, including improved freeze–thaw stability, higher end viscosity, and better enzymatic digestibility. Starch is a very important carbohydrate in many food and non-food applications. In planta modification of starch by genetic engineering has significant economic and environmental benefits as it makes the chemical or physical post-harvest modification obsolete. An amylosucrase from Neisseria polysaccharea fused to a starch-binding domain (SBD) was introduced in two potato genetic backgrounds to synthesize starch granules with altered composition, and thereby to broaden starch applications. Expression of SBD–amylosucrase fusion protein in the amylose-containing potato resulted in starch granules with a rough surface, a twofold increase in median granule size, and altered physico-chemical properties including improved freeze–thaw stability, higher end viscosity, and better enzymatic digestibility. These effects are possibly a result of the physical interaction between amylosucrase and starch granules. The modified larger starches not only have great benefit to the potato starch industry by reducing losses during starch isolation, but also have an advantage in many food applications such as frozen food due to its extremely high freeze–thaw stability.     

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.     

Reduction of starch granule size by expression of an engineered tandem starch-binding domain in potato plants

Granule size is an important parameter when using starch in industrial applications. An artificial tandem repeat of a family 20 starch-binding domain (SBD2) was engineered by two copies of the SBD derived from Bacillus circulans cyclodextrin glycosyltransferase via the Pro-Thr-rich linker peptide from Xyn10A from Cellulomonas fimi. SBD2 and a single SBD were introduced into the amylose-free potato mutant, amf, using appropriate signal sequences. The accumulation of SBD2 into transgenic starch granules was much higher than that of SBD. In a number of transformants, particularly amfSS3, the starch granules were much smaller than in control plants. The amfSS3 mean granule size was 7.8 microm, compared with 15.2 microm in the control, whereas other starch properties were unaltered. This new starch combines the advantage of the high purity of potato starch with that of the small granule size of other crop species, such as cassava, taro and wheat. This starch may find application in the manufacture of biodegradable plastic films. Both genes were also expressed in Escherichia coli and the affinity for soluble starch of the purified recombinant proteins was determined. SBD2 had an approximately 10-fold higher affinity for starch than SBD, indicating that the two appended SBDs act in synergy when binding to their target polysaccharide ligand.     

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.     

Expression of an engineered granule-bound Escherichia coli maltose acetyltransferase in wild-type and amf potato plants

Starch is used in many industrial applications, but often requires chemical derivatization to enhance its properties before use. In particular, the stability of starch polymers in solution is improved by acetylation. A drawback of this treatment is the use of pollutant chemicals. A biological alternative to chemical derivatization was investigated by the expression of an amyloplast-targeted Escherichia coli maltose acetyltransferase ( MAT ) gene in tubers of wild-type (Kardal) and mutant amylose-free ( amf ) potato plants. MAT was expressed as such, or fused to the N- or C-terminus of a non-catalytic starch-binding domain (SBD) to target the starch granule. Starch granules derived from transgenic plants were found to contain acetyl groups, although their content was low, opening up an avenue to move away from the post-harvest chemical derivatization of starch. MAT inside starch granules was found to be active post-harvest when supplied with acetyl-coenzyme A and glucose or maltose, but it did not acetylate starch polymers in vitro . Starch granules from transformants in which MAT alone was expressed also showed MAT activity, indicating that MAT is accumulated in starch granules, and has affinity for starch by itself. Furthermore, starch granule morphology was altered, and fusion proteins containing MAT and SBD seemed to have a higher affinity for starch granules than two appended SBDs. These results are discussed against the background of the quaternary structure of MAT.     

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.     

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).     

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.     

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.     

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and ¹H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.     

Change of granular and molecular structures of waxy maize and potato starches after treated in alcohols with or without hydrochloric acid

Starches from waxy maize and potato were treated in methanol and 2-propanol either with or without 0.36% hydrochloric acid at 65 °C for 1 h. The granule morphology, molecular structure and pasting properties of the starches were determined and the effects of treatments on the granule and molecular structures of starch were investigated. Starch treated in alcohols without acid showed loss of native order through the hilum of granules, and no obvious molecular degradation was found. However, acid–alcohol treated starch showed many cracks inside granules, and both waxy maize and potato starches showed obvious molecular degradation after treated. Furthermore, the amylose chains and long chains of amylopectin of starch were more easily degraded with acid–alcohol treatment. The pasting viscosity of acid–alcohol treated starches were also obviously less than that of their counterpart native starch and starch after alcohol treatment. The extent of degradation of molecules and the decrease of pasting viscosity on potato starch after acid–alcohol treated were more obvious than that of waxy maize starch. The result indicates that the degradation preferentially occur in the amorphous region when starch treated by acid–alcohol, and the degradation of starch molecules enhances the amorphous excretion and the occurrence of cracks inside the granules.     

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.     

Characterization of Molecular Structure of Starch Granules in Suspension-cultured Cells from Ipomoea cordatotriloba Denn.

The molecular structure of starch granules formed in suspension-cultured cells of Ipomoea cordatotriloba Denn. was characterized by its chain length distribution, which was compared to those of the starches from the root and leaf of the original plant. The cultured cell starches were spherical and had a very small granule size (about 2 μm). The debranched starches roughly separated into three fractions during gel-permeation chromatography, and the fractions were defined as Fr.1, 2, and 3, respectively. The chain length distribution of the debranched cultured cell starch showed that the high molecular weight fraction (Fr.1), referred to as an amylose fraction, was much less than those of the root and leaf starches. The ratio of the two lower fractions (Fr.3/Fr.2) of the cultured cell starch, which was mainly derived from unit chains of amylopectin, was greatest among the starches, suggesting that the amylopectin from the cultured cell starch has much shorter unit chains. By X-ray diffraction analysis, it was found that both cultured cell and leaf starch granules have low crystallinity.     

Quantification of total iodine in intact granular starches of different botanical origin exposed to iodine vapor at various water activities

Iodine has been used as an effective tool for studying both the structure and composition of dispersed starch and starch granules. In addition to being employed to assess relative amylose contents for starch samples, it has been used to look at the molecular mobility of the glucose polymers within intact starch granules based on exposure to iodine vapor equilibrated at different water activities. Starches of different botanical origin including corn, high amylose corn, waxy corn, potato, waxy potato, tapioca, wheat, rice, waxy rice, chick pea and mung bean were equilibrated to 0.33, 0.75, 0.97 water activities, exposed to iodine vapor and then absorbance spectra and LAB color were determined. In addition, a new iodine quantification method sensitive to <0.1% iodine (w/w) was employed to measure bound iodine within intact granular starch. Amylose content, particle size distribution of granules, and the density of the starch were also determined to explore whether high levels of long linear glucose chains and the surface area-to-volume ratio were important factors relating to the granular iodine binding. Results showed, in all cases, starches complexed more iodine as water content increased and waxy starches bound less iodine than their normal starch counterparts. However, much more bound iodine could be measured chemically with waxy starches than was expected based on colorimetric determination. Surface area appeared to be a factor as smaller rice and waxy rice starch granules complexed more iodine, while the larger potato and waxy potato granules complexed less than would be expected based on measured amylose contents. Corn, high amylose corn, and wheat, known to have starch granules with extensive surface pores, bound higher levels of iodine suggesting pores and channels may be an important factor giving iodine vapor greater access to bind within the granules. Exposing iodine vapor to moisture-equilibrated native starches is an effective tool to explore starch granule architecture.