Different genetic strategies to generate high amylose starch mutants by engineering the starch biosynthetic pathways

This review systematically documents the major different strategies of generating high-amylose (HAS) starch mutants aiming at providing high resistant starch, by engineering the starch biosynthesis metabolic pathways. We identify three main strategies based on a new representation of the starch structure: 'the building block backbone model': i) suppression of starch synthases for reduction of amylopectin (AP) side-chains; ii) suppression of starch branching enzymes (SBEs) for production of AM-like materials; and iii) suppression of debranching enzymes to restrain the transformation from over-branched pre-AP to more ordered AP. From a biosynthetic perspective, AM generated through the second strategy can be classified into two types: i) normal AM synthesized mainly by regular expression of granule-bound starch synthases, and ii) modified linear AP chains (AM-like material) synthesized by starch synthases due to the suppression of starch branching enzymes. The application of new breeding technologies, especially CRISPR, in the breeding of HAS crops is also reviewed

Production of amylopectin and high-amylose starch in separate potato genotypes

Starch is one of the most important processed products from agriculture. Two main outlets can be identified; starch is either enzymatically processed for the production of sweeteners and as raw material for fermentation or channelled to various applications as dry starch. Native or chemically modified starches are utilized in food as well as non-food applications, where the specific physicochemical properties are main determinants for their respective use. Starch consists of two different molecules, amylose and amylopectin. To be able to take the full benefit of the unique properties of either component it is of interest to divide the production of amylose and amylopectin into separate plant genotypes. In the presented work, potatoes producing either amylopectin or high-amylose starch were achieved using genetic modification. For potato transformation a highly efficient protocol was developed for a herbicide selection gene instead of the commonly used nptII antibiotic selection gene. In order to achieve respective starch qualities, the expression of genes important for amylopectin or amylose synthesis was silenced. Antisense technology as well as the expression of dsRNA was investigated where the expression of dsRNA was determined to be at least ten-fold more efficient for gene silencing. An added benefit of dsRNA expression was that a higher fraction of silenced transgenic lines compared to the use antisense were associated with single copy T-DNA integrations. One amylopectin potato line was furthermore characterized regarding genetic and chemical composition. The T-DNA was found integrated as an inverted repeat with the inverted repeat region extending into potato chromosomal DNA. This transgenic locus was found to be more consistent with integration into a double-stranded chromosomal break than insertion by a mechanism nicking one strand of the locus. The high-amylose trait generally resulted in a higher tuber fresh weight yield, much elevated sugar levels and a decreased starch content. Amylose levels were obtained where very limited amounts of material recognizable as amylopectin could be found. The production of amylopectin and amylose was divided into separate genotypes but additional factors are needed to be able to produce amylose at levels comparable to starch contents of cultivated potatoes

Characterization of the starch branching enzymes in Arabidopsis thaliana

The roles of three starch branching enzyme isoforms, BE1, BE2 and BE3, in Arabidopsis leaf starch metabolism were investigated by AtBE mutant analysis. We also determined the expression patterns of BEs in Arabidopsis by GUS staining and microarray experiments. The possible correlation of BE gene expression with other genes in the Arabidopsis genome was analyzed to identify putative genes that are functionally correlated with BEs. Motifs in the promoters of the Arabidopsis BE genes were identified to better understand their expression differences. BE2 and BE3 expression levels during a diurnal cycle were evaluated with isoform specific antibodies. Meantime, the in vitro biochemical activities of purified recombinant BE2 and BE3 proteins were investigated by three different assays to better understand their specific functions in starch biosynthesis. Analyses of the structures of the final products from the in vitro BE-amylose reactions were done by FACE with two different forms of amylose

Allelic effects on starch structure and properties of six starch biosynthetic genes in a rice recombinant inbred line population

BACKGROUND: The genetic diversity of six starch biosynthetic genes (Wx, SSI, SSIIa, SBEI, SBEIIa and SBEIIb) in indica and japonica rices opens an opportunity to produce a new variety with more favourable grain starch quality. However, there is limited information about the effects of these six gene allele combinations on starch structure and properties. A recombinant inbred line population from a cross between indica and japonica varieties offers opportunities to combine specific alleles of the six genes. RESULTS: The allelic (indica vs japonica) effects of six starch biosynthetic genes on starch structure, functional properties, and abundance of granule bound proteins in rice grains were investigated in a common genetic background using a recombinant inbred line population. The indica Wx (Wxi) allele played a major role while indica SSI (SSIi), japonica SSIIa (SSIIaj) and indica SBEI (SBEIi) alleles had minor roles on the increase of amylose content. SSIIaj and japonica SBEIIb (SBEIIbj) alleles had a major and a minor role on high ratio of ∑DP ≤ 10 to ∑DP ≤ 24 fractions (RCL10/24), respectively. Both major alleles (Wxi and SSIIaj) reduced peak viscosity (PV), onset, peak and end gelatinization temperatures (GTs) of amylopectin, and increased amylose-lipid complex dissociation enthalpy compared with their counterpart-alleles, respectively. SBEIIai and SBEIIbj decreased PV, whereas SSIi and SBEIIbj decreased FV. SBEIi reduced setback viscosity and gelatinization enthalpy. RCL10/24 of chain length distribution in amylopectin is negatively correlated with PV and BD of paste property and GTs of thermal properties. We also report RILs with superior starch properties combining Wxi, SSIj, SSIIaj, SBEIi and SBEIIbj alleles. Additionally, a clear relation is drawn to starch biosynthetic gene alleles, starch structure, properties, and abundance of granule bound starch biosynthetic enzymes inside starch granules. CONCLUSIONS: Rice Wxi and SSIIaj alleles play major roles, while SSIi, SBEIi, SBEIIai and SBEIIbj alleles have minor roles in the determination of starch properties between indica and japonica rice through starch structural modification. The combination of these alleles is a key factor for starch quality improvement in rice breeding programs. RCL10/24 value is critical for starch structure and property determination.Jixun Luo was supported by CSC (Chinese Scholarship Council) and Australian National University scholarships. This work was funded by CSIRO Food Future National Research Flagship

Investigation of the function of branching enzymes I, IIa, and IIb in the determination of amylopectin structure and regulation of starch biosynthesis

Starch is one of the most abundant carbohydrates on earth serving vital roles as the primary energy reserve of plants, a significant source of calories in the human diet, and a renewable resource for other industrial applications. The multiplicity of starch biosynthetic enzymes is evolutionarily conserved in plants suggesting their functions are essential in starch biosynthesis. The overall goal of this research is to determine the specific molecular functions of the multiple starch branching enzyme (BE) isoforms in maize.;This dissertation first addresses what, if any, are the differences in enzymatic properties of BEIIa and BEIIb. Two polyclonal antisera were produced, alphaBEI and alphaBEIIab which specifically identify BEI and both BEII isoforms, respectively. Chromatographic purification techniques separated native BEIIa or BEIIb from the other two branching enzymes. The capacities to modify the structure of amylose were compared between native BEIIa and BEIIb and recombinant BEIIa (rIIa). The evolutionary conservation of BEIIa and BEIIb is not accounted for by their enzymatic differences. Differences in the glucan chain length distribution created by native BEIIa and rIIa suggest that in vivo there are factors affecting the activity of BEs.;This dissertation next addresses are branching enzymes involved in multi-subunit complexes (MSCs)? This research provides direct evidence BEIIa and SSI interact with each other in MSC and that SSIII may be a component of the MSCs. Indirect evidence from GPC analysis of BEIIb and multiple electrophoretic mobility forms of BEI suggests that they may be involved in MSCs. Pleiotropic effects in branching enzyme mutants on starch synthase (SS) activity revealed at least three SS band activities are affected when BEIIa was mutated. Further research is needed to investigate the functional significance of the complexes involving BEIIa, SSI and possibly SSIII.;This dissertation addresses what branching enzyme isoforms exist in leaves as compared to endosperm. The same BEI polypeptide present in maize endosperm was found to be present in maize leaves. Leaf BEI was found to be similar to endosperm BEI in predicted molecular weight, approximately 80 kDa, in molecule size, a monomeric protein, and also possesses multiple electrophoretic mobility forms

Sequence variation and phylogenetic relationship analysis of starch branching enzyme I gene (SBEI) in rice varieties from China, Laos and Thailand

The coding sequence of starch branching enzyme I gene (SBEI) of 30 rice varieties from China, Laos and Thailand were cloned. All thirty sequences contain 2,463 bp and 14 exons and encode for 820 amino acids. Three sites of Single Nucleotide Polymorphisms (SNPs) A < C, T < C, and T < C were found at positions 1,107, 2,156 and 2,271 in Exon with 6, 13 and 14 respectively. The SNPs at position 1,107 A < C and position 2,271 T < C were silent mutations. The SNP at position 2,156 T < C was a missense mutation and induced a mutation from valine (GTG) to alanine (GCG). Three haplotypes A/T/T, C/T/C and C/C/C were observed. The phylogenetic analysis of 81 SBEI CDS sequences, out of which 30 are from this study and 51 are from previous, classifies them into 2 major groups using 4 sequences as outgroup. The group of monocot comprised of rice, barley, wheat, sorghum whereas maize and the group of dicot comprised of potato, cassava, poplar, Chinese chestnut, bean, legumes and apple. The group of rice SBEI CDS was a major clade in monocot group with high bootstrap value. SBEI gene of rice from China, Laos and Thailand, wheat, apple and poplar contain 14 exons while SBEI gene of rice from Japan and Korea contained only 12 exons. The GC content of SBEI gene of rice varieties was lower than that of wheat and apple but higher than that of poplar

Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato

DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.EEA BalcarceFil: Zhao, Xue. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: Jayarathna, Shishanthi. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: Turesson, Helle. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Fält , Ann Sofie. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Nestor, Gustav. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: González, Matías Nicolás. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Olsson, Niklas. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Beganovic, Mirela. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Hofvander, Per. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Andersson, Roger. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: Anderson, Mariette. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia

Novel potato starch : new structure and beneficial qualities

This thesis presents a simplified method for determining the internal molecular structure of whole starch without prior amylopectin isolation. The structure of potato and barley whole starches, the thermal properties of starch from potato lines with different genetic backgrounds and the relationship between molecular structure and functional properties of starch were examined in the thesis. The internal B-chain distribution and building block composition of amylopectin were characterised effectively by degrading starch into β-limit dextrins (β-LDs), α- limit dextrins (α-LDs) and building blocks. Great variations in internal structure were observed for starches from different plant sources and genetic backgrounds. The general composition of intermediate and large building blocks and the proportion of fingerprint B-chains (Bfp-chains), in size order, were determined for starches with decreasing amylose content. Thermal properties (gelatinisation and retrogradation) of potato starches were investigated using differential scanning calorimetry. Amylopectin lines with a high degree of mutations in multiple genes showed a broader gelatinisation temperature range and lower enthalpy of gelatinisation and retrogradation. Various internal structure parameters were found to affect the thermal properties of potato starch. A dense structure of building blocks led to higher gelatinisation temperatures and enthalpy, while retrogradation was found to be favoured by more large building blocks and many short internal chains. The high-amylose potato line T-2012 was shown to have higher levels of resistant starch and dietary fibre than the parental variety after cooking. The level of resistant starch increased further after one extra day of cold storage. T-2012 had a very large fraction of long outer amylopectin chains and intermediate-sized inner amylopectin chains, and more intermediate and large building blocks, than the parental potato. The unique amylopectin structure of T-2012 starch favoured formation of recrystallised amylopectin that did not split as easily as ordinary potato starch and was resistant to enzyme digestion

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed

Plastidial Starch Phosphorylase in Sweet Potato Roots Is Proteolytically Modified by Protein-Protein Interaction with the 20S Proteasome

Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions