An Invertase Inhibitor from Maize Localizes to the Embryo Surrounding Region during Early Kernel Development

Abstract

Invertase activity is thought to play a regulatory role during early kernel development by converting sucrose originating from source leaves into hexoses to support cell division in the endosperm and embryo. Invertases are regulated at the posttranslational level by small protein inhibitors, INVINHs. We found that in maize (Zea mays), an invertase inhibitor homolog (ZM-INVINH1) is expressed early in kernel development, between 4 and 7 d after pollination. Invertase activity is reduced in vitro in the presence of recombinant ZM-INVINH1, and inhibition is attenuated by pre-incubation with sucrose. The presence of a putative signal peptide, fractionation experiments, and ZM-INVINH1::green fluorescent protein fusion experiments indicate that the protein is exported to the apoplast. Moreover, association of ZM-INVINH1 with the glycoprotein fraction by concanavalin A chromatogaphy suggests that ZM-INVINH1 interacts with an apoplastic invertase during early kernel development. ZM-INVINH1 was localized to the embryo surrounding region by in situ analysis, suggesting that this region forms a boundary, compartmentalizing apoplast invertase activity to allow different embryo and endosperm developmental rates.Kernel development in maize (Zea mays) proceeds through a series of tightly regulated, overlapping stages. After double fertilization, during the prestorage phase, two distinct cell types are established: the triploid endosperm and the diploid embryo. Despite clearly different cell fates, the embryo and endosperm both rely upon photosynthate from source leaves transported through the maternal pedicel region of the developing kernel, ending at the terminal phloem cells. The presence of Suc-hydrolyzing enzymes, which produce hexose sugars from Suc, have been identified as critical for the establishment of the prestorage phase of seed development, and Suc hydrolysis is an important component of realizable plant yield (Cheng and Chourney, 1999;Weschke et al., 2003). The "invertase control hypothesis," largely based on work from dicots (Wobus and Weber, 1999), is supported in maize by the presence of a cell wall invertase, INCW2, localized to the basal endosperm transfer layer (Talercio et al., 1999). Mutations in this gene result in miniature kernels (the mn1 mutation) and have a severely reduced endosperm (Cheng et al., 1996;Vilhar et al., 2002). Recently, the association of a IVR2, a soluble invertase expressed during early kernel development, with seed yield under conditions of limiting photosynthesis suggests that soluble invertases also play a significant role in providing hexose sugars to support cell division during the prestorage phase (Andersen et al., 2002), as has been previously suggested (Zinselmeier et al., 1999).Invertases exhibit complex regulation at the transcriptional and posttranscriptional levels in response to developmental, environmental, and carbohydrate signals (Sturm, 1999). In addition, small (Ͻ20-kD) inhibitor proteins (INVINH) have been associated with invertase preparations in a number of dico...

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“…4 and 8A) provides a basis for generating Glc as signals to stimulate endosperm nuclear division but not embryonic cell division. Consistent with this hypothesis is the localization of a CWIN inhibitor at the boundary between the endosperm and embryo in 5-d maize seed (Bate et al, 2004), which could help to minimize Glc flow to the young embryo at this stage. CWIN in the TCP at the innermost area of the seed coat (Fig.…”

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The Development of Endosperm in Grasses

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“…8,9 Such a developmental sequence is of great importance for proper seed development. There is compelling evidence that the endosperm drives seed and embryo growth, 10,11 and controls seed size in a range of species.…”

“…inhibitor at the boundary between the endosperm and embryo in 5-d maize seed, 8 could also help to minimize hexose flow to the young embryo, to ensure nuclear division in endosperm but a quiescent status in embryo at this stage.…”