Genetic evidence that invertase-mediated release of hexoses is critical for appropriate carbon partitioning and normal seed development in maize

Cell wall-bound invertase (CWI) is spatially and temporally the first enzyme which metabolizes the incoming sucrose in developing seed of maize (Zen mays). Our previous studies have shown that the cell wall-bound invertase-2 (INCW2) isozyme encoded by the wild-type gene of the Miniature1 (Mn1) seed locus plays a critical role in seed development. Null mutations of the gene, such as the mn1 seed mutant which lacks invertase activity, are associated with a loss of similar to 70-80% of the normal seed weight. We show here that under in vitro kernel culture conditions the hexose-based medium was similar to the sucrose-based medium in promoting the normal development of kernels of the Mn1, but not of the mutant mn1, genotype. Anatomical, biochemical, and immunohistological data showed that the mn1 kernels retain their mutant phenotype regardless of the presence of sucrose or hexoses in the culture media. The most drastic changes in the mn1 seed mutant were associated with a significant reduction in the size of the endosperm, but not in the pattern or the level of starch localization. Because Mn1 expression was temporally coincident with the endosperm cell divisions, INCW2 must play a critical role in providing hexose sugars for mitotic division, and only a minor role in generating carbon skeletal substrates for starch biosynthesis in the early stages of endosperm development. Furthermore, a lack of the wild-type seed phenotype of the mn1 mutant in hexose media suggests that a metabolic release of hexoses catalyzed by INCW2, rather than an exogenous source, is critical for both generating appropriate sugar-sensing signals for gene expression and for normal endosperm development.