Lignification of developing maize (Zea mays L.) endosperm transfer cells and starchy endosperm cells

Abstract

Endosperm transfer cells in maize have extensive cell wall ingrowths that play a key role in kernel development. Although the incorporation of lignin would support this process, its presence in these structures has not been reported in previous studies. We used potassium permanganate staining combined with transmission electron microscopy - energy dispersive X-ray spectrometry as well as acriflavine staining combined with confocal laser scanning microscopy to determine whether the most basal endosperm transfer cells (MBETCs) contain lignified cell walls, using starchy endosperm cells for comparison. We investigated the lignin content of ultrathin sections of MBETCs treated with hydrogen peroxide. The lignin content of transfer and starchy cell walls was also determined by the acetyl bromide method. Finally, the relationship between cell wall lignification and MBETC growth/flange ingrowth orientation was evaluated. MBETC walls and ingrowths contained lignin throughout the period of cell growth we monitored. The same was true of the starchy cells, but those underwent an even more extensive growth period than the transfer cells. Both the reticulate and flange ingrowths were also lignified early in development. The significance of the lignification of maize endosperm cell walls is discussed in terms of its impact on cell growth and flange ingrowth orientation.

Keywords: cell growth analysis; flange ingrowths; lignin; maize endosperm; reticulate ingrowths; starchy cells; transfer cells.

Figure 1

Images obtained by TEM-EDS (A–E) and TEM (F) of samples stained with KMnO₄. Polygons represent the boundaries of EDS readings in each sample. (G–J) CLSM images of acriflavine-stained samples. (A) MBETCs at 12 DAP – readings were obtained from the reticulate ingrowths. (B) MBETCs at 12 DAP – readings were obtained from vesicles adjacent to reticulate ingrowths. (C) MBETCs at 12 DAP – readings were obtained from anticlinal walls. (D) MBETCs at 20 DAP – readings were obtained from flange ingrowths and inner periclinal walls (^*). (E) Starchy cells at 20 DAP – readings were obtained from the walls. (F) MBETCs at 6 DAP – readings were obtained from vesicles releasing their contents into reticulate ingrowths. (G) MBETCs at 9 DAP. (H) Starchy cells at 9 DAP. (I) MBETCs at 25 DAP. (J) Starchy cells at 25 DAP. AW, anticlinal wall; FI, flange ingrowth; OPW, outer periclinal wall; RI, reticulate ingrowth. Scale bars: (A–F) = 1 μm; (G–J) = 20 μm.

Figure 2

TEM images of ultrathin MBETC sections at 10 DAP, after H₂O₂ treatment without contrast (A and B) and control sections at 6 DAP without treatment and contrast (C). (A) After a 15-min H₂O₂ treatment, some vesicles lacked electron density (black arrows), whereas reticulate ingrowths and the OPW show regions with less electron density (white arrows). (B) After a 60-min H₂O₂ treatment, many vesicles lost their electron density (black arrows), whereas reticulate and flange ingrowths, the OPW and anticlinal walls reveal large regions with less electron density (white arrows). (C) Control sections with electron-dense vesicles (black arrows), whereas the walls and flange ingrowths have a weak electron density. AW, anticlinal wall; FI, flange ingrowth; NUC, nucleus; OPW, outer periclinal wall; RI, reticulate ingrowth. Scale bars: (A,B) = 2 μm, (C) = 1 μm.

Figure 3

(A) Growth analysis of MBETCs, showing cell areas (μm²) of developing kernels (4–20 DAP, equivalent to 62.0–300.2 GDD) and adjusted growth curve (cell area = 903.04/(1 + exp(15.81 − 0.15 × GDD)^∧(1/4.37)), R² = 67.15%). (B) Growth analysis of starchy cells, showing cell areas (μm²) of developing kernels (5–35 DAP, equivalent to 73.5–522.0 GDD) and adjusted growth curve (cell area = −43582.6 + 10195.3 × ln(GDD), R² = 92.3%). (C) Flange ingrowth angles (in relation to the nearest anticlinal wall) of developing MBETCs (6, 12, and 20 DAP, and an average of the three sampling dates).