Non‐GMO potato lines, synthesizing increased amylose and resistant starch, are mainly deficient in isoamylase debranching enzyme

Abstract

Summary Solanum tuberosum potato lines with high amylose content were generated by crossing with the wild potato species Solanum sandemanii followed by repeated backcrossing to Solanum tuberosum lines. The trait, termed increased amylose (IAm), was recessive and present after three generations of backcrossing into S. tuberosum lines (6.25% S. sandemanii genes). The tubers of these lines were small, elongated and irregular with small and misshaped starch granules and high sugar content. Additional backcrossing resulted in less irregular tuber morphology, increased starch content (4.3%–9.5%) and increased amylose content (29%–37.9%) but indifferent sugar content. The amylose in the IAm starch granules was mainly located in peripheral spots, and large cavities were found in the granules. Starch pasting was suppressed, and the digestion‐resistant starch (RS) content was increased. Comprehensive microarray polymer profiling (CoMPP) analysis revealed specific alterations of major pectic and glycoprotein cell wall components. This complex phenotype led us to search for candidate IAm genes exploiting its recessive trait. Hence, we sequenced genomic DNA of a pool of IAm lines, identified SNPs genome wide against the draft genome sequence of potato and searched for regions of decreased heterozygosity. Three regions, located on chromosomes 3, 7 and 10, respectively, displayed markedly less heterozygosity than average. The only credible starch metabolism‐related gene found in these regions encoded the isoamylase‐type debranching enzyme Stisa1. Decreased expression of mRNA (>500 fold) and reduced enzyme activity (virtually absent from IAm lines) supported Stisa1 as a candidate gene for IAm.

Expression Pattern of Starch Biosynthesis Genes in Relation to the Starch Molecular Structure in High-Amylose Maize

“…SBEI seems to prefer amylose as a substrate and, as a consequence, suppression of SBEI increased the relative amylose content. ISAII suppression can cause high amylose content by decreasing the amylopectin content, e.g., as demonstrated, a potato mutant deficient in this enzyme …”

Interfacial Catalysis during Amylolytic Degradation of Starch Granules: Current Understanding and Kinetic Approaches

“…Previous studies have proposed that, for high amylose maize starches, amylose is more concentrated at the periphery than the core, while the opposite is observed for normal and waxy starches [54]. However, such AM distributions can be complex depending on genotypes and mutations in the genes coding for target starch metabolizing enzymes [55][56][57]. Such knowledge, such as the degree of branching and chain length distribution on the surface, is crucial for starch hydrolysis, as the flexible glucan chains on the granule surface are considered a primary factor influencing the susceptibility of starch to amylolysis [58].…”

“…Enzymatic hydrolysis of starch granules is a complex and heterogeneous catalytic process [64] that depends on the interplay among the widely different granule surfaces and matrix structures described above, the substrate recognition and catalysis by the hydrolase. Importantly, at the granular starch surface, a fraction of glucan chains may be accessible, which potentially provides efficient binding sites and substrates for starch-active enzymes [56,57]. The rate of hydrolysis can be controlled by three main factors: (i) diffusion of the enzyme toward the granule surface; (ii) adsorption onto structures at the granule surface; and (iii) catalytic glycoside bond hydrolysis (Figure 3).…”

Amylose in starch: towards an understanding of biosynthesis, structure and function

“…Two factors lead to the increased amylose content in these mutants: First, as BE is required for amylopectin biosynthesis, suppressing its activity reduces the amount of amylopectin and increases the relative proportion of amylose. Similarly, many mutations in the amylopectin biosynthesis pathway have been observed to increase amylose content in Arabidopsis leaves and various crops: including other starch synthases (Wang et al ., 1993; Delvallé et al ., 2005; Fujita et al ., 2007; Zhang et al ., 2008; Szydlowski et al ., 2009), and isoamylase (Dauvillée et al ., 2001; Tziotis et al ., 2004; Blennow et al ., 2020). Second, the suppression of BE reduces the frequency of branching on amylopectin, resulting in the formation of long amylose‐like chains on amylopectin.…”