Electron microscopy analysis of maize basal endosperm transfer cells processed by high-pressure freezing and freeze-substitution

As heterotrophs, developing maize seeds are dependent on mother plant for their carbon and nitrogen needs. Upon symplastic transport to phloem termini in pedicel and a short-distance postphloem transit, photoassimilates are unloaded into growing maize seed by the basal endosperm transfer cells (BETCs). A ubiquitous feature of all transfer cells is labyrinth wall, the wall-ingrowth (WIG), which increases the plasma membrane area and is believed to confer greater maternal-to-filial transport capacity. To investigate intracellular organization of the maize BETC, we performed electron microscopy analysis of maize BETCs preserved by high-pressure freezing and freeze-substitution. These two advanced sample preparation techniques have not been used for imaging maize BETCs, yet. Maize kernels at 7 days after pollination (DAP) and 12 DAP were dissected and cryofixed with Bal-tec HPM010. The samples were freeze-substituted in 2% OsO4 in acetone and embedded in EPON resin. Figure 1 and 2 show longitudinal sections of a 7 DAP BETC and a 12 DAP BETC, respectively. The most abundant organelles in the mature BETCs are mitochondria and the ER (Fig. 1A and 2A). In the 7 DAP BETCs, Golgi stacks (Fig. 1B) and multivesicular bodies (MVBs) (Fig. 1C) are readily identified. In vicinity of the basal cell wall, secretory-type vesicles that fuse with the plasma membrane were seen (Fig. 1D). Golgi stacks and MVBs are rare in 12 DAP BETCs. In BETCs, mitochondria are abundant and mostly spherical (diameters 300~600 nm). Interestingly, in 12 DAP BETCs, the mitochondria accumulate in the basal half (85.5%) of the cells and frequently dispersed in the interstices of WIGs as reported previously. [1-2] (Fig. 2A and 2B). It is likely that the WIG-associated mitochondria generate ATP to support active uptake of nutrients. In the 7 DAP BETC, mitochondria begin to locate in the basal region prior to WIG assembly suggesting that mitochondria are actively transferred to the future WIG sites (67.3%, Fig. 1A). The ER tubules/cisternae permeate rest of the cytoplasm but accumulates more on the apical side of the cell (Fig. 2A). The sidewall and apical wall of the 12 DAP BETC have numerous plasmodesmata forming a symplastic domain with neighboring BETCs and endosperm cells (Fig 2C and 2D). As is well known, no symplastic connections via plasmodesmata were found in the WIG facing the maternal pedicel [3]. The cell wall composition is not uniform in WIG as evidenced by black blotches dispersed in both young and old WIGs (Fig 2B and 2C). We plan to characterize secretory and endocytic organelles as well as cytoskeleton systems that contribute to WIG formation. We will also carry out immunogold localization of known BETCspecific proteins including INCW2, a cell wall invertase to gain insight into functional organization of the BETC.