Miniature1-encoded cell wall invertase is essential for assembly and function of wall-in-growth in the maize endosperm transfer cell

Abstract

The miniature1 (mn1) seed phenotype in maize (Zea mays) is due to a loss-of-function mutation at the Mn1 locus that encodes a cell wall invertase (INCW2) that localizes exclusively to the basal endosperm transfer cells (BETCs) of developing seeds. A common feature of all transfer cells is the labyrinth-like wall-in-growth (WIG) that increases the plasma membrane area, thereby enhancing transport capacity in these cells. To better understand WIG formation and roles of INCW2 in the BETC development, we examined wild-type and mn1 mutant developing kernels by cryofixation and electron microscopy. In Mn1 seeds, WIGs developed uniformly in the BETC layer during 7 to 17 d after pollination, and the secretory/endocytic organelles proliferated in the BETCs. Mitochondria accumulated in the vicinity of WIGs, suggesting a functional link between them. In the mn1 BETCs, WIGs were stunted and their endoplasmic reticulum was swollen; Golgi density in the mutant BETCs was 51% of the Mn1 Golgi density. However, the polarized distribution of mitochondria was not affected. INCW2-specific immunogold particles were detected in WIGs, the endoplasmic reticulum, Golgi stacks, and the trans-Golgi network in the Mn1 BETCs, while immunogold particles were extremely rare in the mutant BETCs. Levels of WIG development in the empty pericarp4 mutant was heterogeneous among BETCs, and INCW2 immunogold particles were approximately four times more abundant in the larger WIGs than in the stunted WIGs. These results indicate that polarized secretion is activated during WIG formation and that INCW2 is required for normal development of WIGs to which INCW2 is localized.

Figure 1.

Cell wall ingrowth formation is retarded in the mn1-1 mutant BETCs. Toluidine blue-stained semithin sections of wild-type (Mn1) BETL (A–C) at 7, 12, and 17 DAP and mn1-1 mutant BETL (D–F) at 12 and 17 DAP are shown. A, WIGs are not observed in Mn1 BETL at 7 DAP. B, At 12 DAP, WIGs at the basal side of the BETL are clearly discerned (asterisks). C, Long and complex WIGs at 17 DAP (double arrow). D, WIGs are not detected in 12-DAP mn1-1 BETL, except for some patchy WIGs (dashed circle). E and F, WIGs in 17-DAP mn1-1 BETL (asterisks in E). They are smaller (double arrow in F) than Mn1 17-DAP WIGs (double arrow in C). Bars = 20 μm.

Figure 2.

Electron microscopy analysis of Mn1 BETCs. Longitudinal sections of 7-DAP (A, B, and L), 12-DAP (C–G and M), and 17-DAP (H–K and N) BETC samples preserved by high-pressure freezing. A and B, BETCs at 7 DAP. Mitochondria are seen adjacent to the basal side (dashed ovals in A and arrowheads in B). WIG initials are marked with arrows in B. C and D, BETCs at 12 DAP. WIGs consist of a fibrous meshwork (circle in D) and dark blotches (dashed circle in D). Mitochondria (arrowheads in D) are often located in the interstices of the WIGs. E, Golgi stacks (arrowheads), TGN cisternae, and multivesicular bodies (arrow) are abundant in 12-DAP BETCs. A mitochondrion is denoted with an asterisk. F, Tubulovesicular TGN compartments (dashed ovals) in 12-DAP BETCs. G, Higher magnification of a 12-DAP TGN compartment. H, BETC at 17 DAP. I, Cortical cytoplasm of a 17-DAP BETC showing WIGs, mitochondria (asterisks), and ER (arrowheads). J and K, Golgi stacks, ER, vacuoles, and WIGs in 17-DAP BETCs. Vesicles accumulate (dashed ovals) adjacent to the Golgi stacks. L to N, Electron micrographs illustrating the distribution of mitochondria in 7-DAP (L), 12-DAP (M), and 17-DAP (N) BETCs. Mitochondria in A, C, and H are highlighted in red in these panels. G, Golgi stack; N, nucleus; PCW, primary cell wall; V, vacuole. Bars = 5 μm in A, C, and H. Bars = 500 nm in B, D to G, and I to K.

Figure 3.

Electron microscopy analysis of mn1-1 BETCs. A to C, mn1-1 BETCs at 12 DAP. A, mn1-1 BETC with WIGs are less convoluted than wild-type WIGs. B, mn1-1 BETC devoid of WIG. C, Higher magnification image of WIGs and peripheral cytoplasm of a mn1-1 BETC. Mitochondria (arrowheads) and swollen ER (asterisks) are marked in A to C. D, mn1-1 BETC at 17 DAP. The large ER cisternae are denoted with dashed ovals. E, Higher magnification image of the peripheral cytoplasm in a 17-DAP mn1-1 BETC. Mitochondria are marked with arrowheads. F, Higher magnification image of the abnormal ER cisternae. Multivesicular bodies are marked with arrows. G and H, Golgi stacks in 12-DAP (G) and 17-DAP (H) mn1-1 BETCs. G, Golgi stack; N, nucleus; PCW, primary cell wall. Bars = 2 μm in A and B, 500 nm in C and E to H, and 5 μm in D.

Figure 4.

Immunogold labeling of INCW2 in Mn1 and mn1-1 BETCs. A, Immunogold labeling of INCW2 in 17-DAP Mn1 BETCs. INCW2 localizes to the WIG. B and C, INCW2-specific immunogold particles are seen in the ER (arrows), Golgi stack (arrowheads), and TGN (arrowheads). The cis- and trans-sides of the Golgi stacks are indicated in C. D, WIG localization of INCW2 in a 10-DAP Mn1 BETC. E, Lack of INCW2-specific immunogold particles in a 17-DAP mn1-1 mutant BETC. The abnormal ER cisternae distinctive of mn1-1 mutant BETCs are indicated. G, Golgi stack; M, mitochondrion. Bars = 1 μm in A and 500 nm in B to E.

Figure 5.

Localization of INCW2 in the WIG in Mn1 and emp4 BETCs. A and B, Immunogold labeling of INCW2 in the Mn1 15-DAP BETCs showing that INCW2 localizes to the WIG but not to the primary cell wall (PCW). A is from a basal cell wall, and B is from a side wall. C, Toluidine blue-stained thin section of 15-DAP emp4 mutant BETCs. W and W′ denote two BETCs, one with sparse WIG and the other with dense WIG, respectively. C is a gray-scale image of a bright-field micrograph. D, Immunogold labeling of INCW2 in two neighboring emp4 BETCs with differing amounts of WIG formation (arrows). INCW2 immunogold particles are more abundant in the thick WIG (WIG2 in W′ cell) than in the thin WIG (WIG1 in W cell). The cell boundary (dashed line) was determined by the position of the primary cell wall. M, Mitochondrion. Bars = 2 μm.

Figure 6.

Golgi densities in the BETC cytoplasm and INCW2 immunogold particle densities in the WIGs. A, Average Golgi densities (number of Golgi stacks μm⁻² cytoplasm in thin sections) in Mn1 (wild type [WT]) BETCs and mn1-1 BETCs at 7, 12, and 17 DAP. The density in 7-DAP mn1-1 BETCs was not determined. B, Average INCW2 gold particle densities in the WIGs of Mn1 (wild type), thick WIGs in emp4, and thin WIGs in emp4 BETCs.