Genetically Modified Starch

State of Art and Perspectives

Ahmed Regina1,2; Zhongyi Li1,2; Matthew K. Morell1,2; Stephen A. Jobling1,2    1 CSIRO Food Futures National Research Flagship, GPO Box 1600, Canberra, Australia
2 CSIRO Plant Industry, GPO Box 1600, Canberra, Australia

Abstract

Shortly after genetic modification of plants became possible in the early 1980’s, attempts were made to modify starch structure by altering the expression of starch biosynthetic genes or by introducing novel enzymes from other organisms. Much of this work was done initially in potato and later work focussed on the model plant Arabidopsis and the regulation of starch breakdown in leaves. In this chapter, we will review recent advances in generating novel starch compositions and structures using new breeding technologies, focusing mainly on cereal starches but including other starches where appropriate advances have been made. We will first describe attempts to alter starch structure in order to improve starch functionality, then explain how increasing knowledge of the regulation of starch biosynthesis is being used to increase starch production, and finish by summarizing new methods for increasing the genetic diversity in crops as well as methods for fine-tuning gene expression in plants in order to bring improved starch-based products with value-added consumer benefits to the marketplace.

Keywords

starch modification

resistant starch

nutrition

amylose

amylopectin

cereal

wheat

endosperm

granule

1 Introduction

Starch is commercially isolated from a wide range of sources including cereal grains such as corn, wheat, rice, and sorghum; roots and tubers such as potato, sweet potato, cassava, and arrowroot; and stem and pith such as sago. The composition of naturally occurring starch is more or less universal with a major component of amylopectin (~ 75%) and a minor component of amylose (~ 25%) irrespective of its source. Both amylose and amylopectin are polymers of α 1,4-linked glucan chains with varying proportions of α(1,6)-linked branch points. While amylose is predominantly linear with less than 1% α(1,6) linkages, amylopectin is a much larger and highly branched molecule with 4-5% α(1,6) linkages.

Starch modification implies efforts to both achieve increased starch production and modify the composition and component structure to impart specific properties to suit particular end uses. The most widely referred target for starch modification is the amylose-amylopectin ratio. Several plants have been modified in their starch biosynthetic pathway to yield both high-amylose and high-amylopectin starches. Both these type of starches have distinct end uses, for example, high-amylopectin starches are used in paper manufacture and in some specialized adhesive applications such as sticky labels on glass jars where the antiwetting properties of the starch prevent the label from peeling in damp conditions. Waxy starches are also widely used in food preparation as they make highly transparent gels that have improved freeze-thaw characteristics compared to normal amylose-containing starch gels. High-amylose starches are also used in adhesive manufacture as the long linear chains of amylose associate strongly together quickly on cooling, making them very useful in high speed applications such as corrugated cardboard production. These starches are also used in specialized food applications such as improving crispiness of coatings during frying, and they have general utility as thickeners and gelling agents (Jobling, 2004). More importantly, the nutritive value of high-amylose starches as a source of resistant starch, a special type of dietary fiber, is being increasingly recognized in the food industry, and this will be discussed in more detail later.

In this chapter, we will review recent advances in generating novel starch compositions and structures using new breeding technologies, focusing mainly on cereal starches but including other starches where appropriate advances have been made. We will first describe attempts to alter starch structure in order to improve starch functionality, then explain how increasing knowledge of the regulation of starch biosynthesis is being used to increase starch production, and finish by summarizing new methods for increasing the genetic diversity in crops as well as methods for fine-tuning gene expression in plants in order to bring improved starch-based products with value-added consumer benefits to the marketplace.

2 Modification of Starch Quality

Starch quality implies the net outcome of a combination of structural features such as the relative proportion of amylose and amylopectin, chain length distribution and branching frequency of these two components, molecular organization and higher-order structure, granule shape and size, lipid and protein content, and covalent modification such as phosphorylation. Our current knowledge suggests interdependence of these features to some extent. The physicochemical properties of starch are highly dependent on the structural features, which in turn influence its behavior while processing. The last few decades have seen successful attempts in altering the amylose content of starch in economically important crops, either toward lowering or toward elevating the amylose compared to the wild-type levels.

3 Starch Modification to Lower Amylose Content

It is now commonplace knowledge that the absence or inactivation of the granule-bound starch synthase 1 gene (GBSS1) encoded by the waxy (Wx) locus in plants gives amylose-reduced or amylose-free phenotype in storage tissues with no apparent reduction in total starch content. The level of reduction in amylose content varies depending on the nature of mutation in the GBSS1 gene. The first reported waxy hexaploid wheat had an amylose content of > 1% (Nakamura, Yamamori, Hirano, Hidaka, & Nagamine, 1995) and was a combination of three null waxy alleles, the Wx-A1 bearing a 23-bp deletion and a 4-bp insertion; the Wx-B1 allele, in which the entire coding region is deleted; and the Wx-D1 allele, which has a 588-bp deletion and a12-bp insertion (Vrinten, Nakamura, & Yamamori, 1999). Identification of variant alleles of Wx genes in each of the three waxy loci (six different alleles identified thus far each on Wx-A1 and Wx-B1 loci and seven alleles on Wx-D1 locus) allowed the generation of grains with a range of phenotypes having 0-30% amylose content (Yasui, 2006; Yasui & Ashida, 2011). In diploid barley, low-amylose waxy cultivars appear to have a 413-bp deletion in the promoter and 5′-untranslated region (UTR) of the GBSS1 gene, whereas an amylose-free barley gene carried an A to T substitution leading to the substitution of an aliphatic amino acid (Val) for an acidic amino acid (Asp) (Patron et al., 2002) In rice, a G/T polymorphism at the 5′-leader/first intron splicing site appeared to regulate the production of mature GBSS mRNA, which in turn influenced the amylose content. The presence of a G at the splicing site promotes normal splicing resulting in enhanced GBSS activity and high amylose content in indica rice compared to japonica rice, in which the presence of T at the junction results in cryptic splicing, leading to lesser GBSS efficiency and lower amylose levels (Cai, Wang, Xing, Zhang, & Hong, 1998; Ni et al., 2011; Wang et al., 1990). This in combination with two other single-nucleotide polymorphisms (SNPs) in GBSS1, an A/C SNP in exon 6 and a C/T SNP in exon 10, accounted for approximately 90% variation in amylose in two separate US and European germplasm collections (Dobo, Ayres, Walker, & Park, 2010).

Silencing of GBSS1 using an RNA interference (RNAi) construct containing 3′-UTR region led to reduced amylose content ranging from 5.9% to 9.0% in transgenic rice lines compared to 17.7-18.0% in wild type (Park et al., 2010).

The most common waxy starch in use currently is from corn or maize, but the range of waxy starches on the market has increased recently with the introduction of amylose-free potato starches into Europe. AVEBE has developed a waxy potato variety Eliane™ using traditional mutation breeding techniques, while BASF has developed Amflora, a GM potato variety through silencing the GBSS1 gene. The cultivation, processing, and industrial use of this GM potato were approved by the European Commission on 2 March 2010. Both of these potatoes are produced in a closed-loop system where seed production, growth, and supply to the processing factory are tightly controlled. Waxy potato starches have much improved functionality giving very clear gels with high freeze-thaw stability compared to normal potato starch. Even further increases in freeze-thaw stability are possible as was demonstrated in a triple waxy/SSII/SSIII downregulated transgenic potato line (Jobling, Westcott, Tayal, Jeffcoat, & Schwall, 2002). Starch from this line had extremely short-chain amylopectin and no amylose, and this reduced syneresis levels to virtually zero even after several freeze-thaw cycles.

4 Starch Modification...