Abstract
The major component of starch is the branched glucan amylopectin. Structural features of amylopectin, such as the branching pattern and the chain length distribution, are thought to be key factors that enable it to form semicrystalline starch granules. We varied both structural parameters by creating Arabidopsis (Arabidopsis thaliana) mutants lacking combinations of starch synthases (SSs) SS1, SS2, and SS3 (to vary chain lengths) and the debranching enzyme ISOAMYLASE1-ISOAMYLASE2 (ISA; to alter branching pattern). The isa mutant accumulates primarily phytoglycogen in leaf mesophyll cells, with only small amounts of starch in other cell types (epidermis and bundle sheath cells). This balance can be significantly shifted by mutating different SSs. Mutation of SS1 promoted starch synthesis, restoring granules in mesophyll cell plastids. Mutation of SS2 decreased starch synthesis, abolishing granules in epidermal and bundle sheath cells. Thus, the types of SSs present affect the crystallinity and thus the solubility of the glucans made, compensating for or compounding the effects of an aberrant branching pattern. Interestingly, ss2 mutant plants contained small amounts of phytoglycogen in addition to aberrant starch. Likewise, ss2ss3 plants contained phytoglycogen, but were almost devoid of glucan despite retaining other SS isoforms. Surprisingly, glucan production was restored in the ss2ss3isa triple mutants, indicating that SS activity in ss2ss3 per se is not limiting but that the isoamylase suppresses glucan accumulation. We conclude that loss of only SSs can cause phytoglycogen production. This is readily degraded by isoamylase and other enzymes so it does not accumulate and was previously unnoticed.
Highlights
The major component of starch is the branched glucan amylopectin
Our results indicate that this phenomenon is largely masked by the presence of ISA1-ISA2, which degrades the aberrant glucan instead of trimming it to amylopectin
Despite the importance of ISA, the phytoglycogen-accumulating phenotype was shown to be incomplete in many cases
Summary
The major component of starch is the branched glucan amylopectin. Structural features of amylopectin, such as the branching pattern and the chain length distribution, are thought to be key factors that enable it to form semicrystalline starch granules. Amylopectin from SS1-deficient mutants of Arabidopsis (Delvallé et al, 2005; Szydlowski et al, 2011) and rice (Oryza sativa; Fujita et al, 2006) has fewer chains with a degree of polymerization (DP; i.e. chain length) between 8 and 12 and more chains with a DP between 17 and 20 compared with the wild-type starches This is consistent with in vitro data for the maize (Zea mays; Commuri and Keeling, 2001) and rice SSI enzymes (Fujita et al, 2006), which preferentially elongate short chains of DP 6 or 7 up to a length of DP 10. Arabidopsis ss mutants have just one round starch granule per chloroplast rather than five or more lenticular granules observed in the wild type
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