Identification of the Maize Amyloplast Stromal 112-kD Protein as a Plastidic Starch Phosphorylase,

Abstract

Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.

Mutation of the Plastidial α-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

“…Although exhibiting a vitreous appearance much like T65, pseudonormal seeds were the amyloplasts. A stroma location was also observed for maize endosperm Pho1 (Yu et al, 2001). …”

“…Pho1 is localized in amyloplasts, the site of starch synthesis (Yu et al, 2001). Several starch biosynthetic enzymes, such as starch synthase I (SSI), are localized in the amyloplast stroma and are also associated with starch (Fujita et al, 2006).…”

“…The two Pho types differ from each other not only in structure and kinetic properties but also in their expression pattern and subcellular localization (Steup, 1990). The plastidial isoform, referred to as Pho1 or Pho-L, has an extra 80-amino acid insertion not present in Pho2 and possesses a high affinity toward low molecular weight linear malto-oligosaccharides (MOS) and amylose, whereas the cytosolic isoform, Pho2 or Pho-H, has a high affinity toward highly branched polyglucans, such as glycogen (Shimomura et al, 1982;Steup, 1988;Yu et al, 2001).…”

Gradually Decreasing Starch Branching Enzyme Expression Is Responsible for the Formation of Heterogeneous Starch Granules

“…Amylopectin is synthesized by the combined action of several isoforms, including starch synthases (SSs), starch branching synthases (SBEs), and debranching enzymes (DBEs; Nakamura, 2002;Davis et al, 2003;Tetlow, 2006). In addition, starch phosphorylase 1 (Pho1) also has been reported to play a role in starch synthesis, although its precise role remains elusive (Yu et al, 2001;Satoh et al, 2008).…”

Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm

“…Plants possess two types of SP, Pho1 and Pho2, localized to the plastid and cytosol, respectively. A number of studies have found that Pho1 gene expression and activity correlate with other starch biosynthetic enzymes, like SS and SBE, suggesting a role in contributing to starch biosynthesis [122,123]. Rice pho1 mutant shows an accumulation of smaller starch granules and abnormal endosperm phenotype [124], and the amylopectin showed a higher proportion of short chains of DP < 11 and a lower proportion of intermediate chains of DP 13-21.…”