Characterization of a alkaline/neutral invertase, ZmINVAN8, in heterotrophic plastids of developing maize endosperm

In young developing sink tissues of plants such as maize (Zea mays L.) endosperm, cellular sucrose (Suc) cleavage is dominated by the invertase (INV) enzyme class whose members comprise a large gene family. This study concerns the alkaline/neutral-INV (A/N-INV) subfamily during the early stages of maize endosperm development (4-14 days after pollination [DAP]), before the start of grain filling. Nine of the ten A/N-INV genes show relatively high expression at early stages of endosperm development, one among which, ZmINVAN8, is the focus of the present study. We determined the subcellular localization of ZmINVAN8 and describe its biochemical characterization. Proteome and marker enzyme analyses of leucoplasts isolated from maize endosperm at 6-10 DAP showed that this A/N-INV isoform is localized to the stroma of heterotrophic plastids. Furthermore, confocal laser scanning microscopic analysis of transiently expressed GFP-tagged ZmINVAN8 in tobacco leaf cells revealed that the putative 62-amino acid transit peptide of the full-length ZmINVAN8 protein (60.94 kDa) is necessary for import into plastids and that this A/N-INV isoform is localized to the stroma. The possible biological role of this Suc-hydrolyzing enzyme inside heterotrophic plastids of the endosperm is discussed.