The role of pullulanase and SSIII in the formation of the vitreous endosperm phenotype in Quality Protein Maize.

Despite being a major food source, maize does not provide a balanced source of protein, because maize lacks some essential amino acids, such as lysine and tryptophan. opaque-2 (o2) mutants highly increased the lysine content, but they were not suitable for commercial development, due to their soft and chalky endosperm. Modifier genes were introgressed into o2 maize that restored hard and vitreous endosperms, while maintaining the high lysine content and these lines were called Quality Protein Maize (QPM). In this study two candidate factors associated with the formation of the vitreous endosperm phenotype in QPM were characterized; Zpu1, encoding pullulanase-type starch debranching enzyme and SSIII, encoding Starch Synthase III. The data showed that the QPM inbred line, K0326Y, had higher pullulanase activity and SSIII abundance than W64Ao2. Recombinant inbred lines (RILs) generated from a W64Ao2 X K0326Y cross demonstrated that kernel vitreousness was positively correlated with pullulanase activity, indicating pullulanase may be one of the key factors affecting vitreousness. Pullulanase activity was highly dependent on Zpu1 alleles and SSIII alleles, indicating that SSIII might be an indirect factor associated with kernel vitreousness that modulates pullulanase activity. Structural analysis of starch isolated from endosperms of RILs showed that the starch fine structure could be altered by changes of pullulanase activity and SSIII abundance. Sequence analysis showed a single amino acid change between the Zpu1 genes derived from W64Ao2 and K0326Y. Therefore, they were cloned and recombinant proteins were used to assay enzyme activity. Pullulanase from W64Ao2 had higher activity than the K0326Y counterpart, which contradicted the native pullulanase activity comparison between W64Ao2 and K0326Y in crude endosperm extracts. Therefore, the null mutants Zpu1-204 and du1-M4 (null mutant of SSIII) were analyzed to identify possible physical or functional interaction between the two enzymes. This showed that pullulanase activity was significantly reduced in du1-M4, which also affected the thermal properties and surface characteristics of starch granules. Thus SSIII may affect the activity of other enzymes in starch biosynthetic pathway, such as pullulanase. These experiments demonstrated that pullulanase and SSIII play a role in the formation of vitreous endosperm in QPM.