PMC

Before the tapetum is isolated, an anther contains endothecium, tapetum, and microspores (a). Microspores were released by vortexing (b). After tapetum isolation, the tapetum and microspores were removed, and the endothecium had integrity (c). The expression of SCR-60 was detected in the isolated tapetum of S-60 homozygotes (d). The DNA methylation state of the SCR-60 promoter in the tapetum of S-22/S-60 heterozygotes was analyzed using the tapetum isolated by our method (e). The thick line under the DNA methylation profile indicates the region homologous to Smi. E endothecium, T tapetum, M microspores, G genomic DNA

Transmission electron microscopy of anther tapetum cells during microgametogenesis in the MicroTom wild type and the slcer6 mutant. A, Wild-type tapetum at the stage of early microspore. The tapetum cell is irregularly shaped, and electron-dense cytoplasm contains mitochondria (M), vacuoles (V), and other organelles. The tapetal wall (TW) facing the locule is lined with electron-dense orbicules (Or). B, slcer6 tapetum at the stage of early microspore. The tapetal cell is almost devoid of cytoplasm. C, Wild-type tapetum at the vacuolate microspore stage. Cytoplasm becomes more disintegrated and contains spheroid lipid drops (arrowheads). D, slcer6 inner tapetum cell with irregularly shaped prominent bodies (asterisks) at the vacuolate microspore stage. E, Outer tapetum at the vacuolate microspore stage in slcer6. The tapetum cell lined with orbicules along the wall has almost no cytoplasm. F, Cross section of a slcer6 anther with microspores (MS). The outer tapetum (OtT) is located at the side of contacting anthers. The inner tapetum (InT) is located closer to the connective tissue. G, Wild-type tapetum at anthesis and mature pollen grain (PG) with highly structured electron-dense cytoplasm. The tapetal wall facing the locule is appressed to that of the middle layer. H, slcer6 tapetum and degenerated pollen grain (DP) at anthesis.

Transmission electron microscopy scans of tapetum morphology in “ZH” and “JX.” (A) Normal tapetum; (B) vacuolized tapetum; (C) degraded tapetum; (D) swollen tapetum; (E) completely degraded tapetum; (F) delayed tapetum degradation. T, tapetum; V, vacuole; P, peroxisome.

(A, B) Tapetum binucleation is gradual (arrows, binucleate tapetum cells; arrowheads, uninucleate tapetum cells). (A) An 1800 μm anther in optical section with a partially binucleate tapetum. (B) An older anther (2200 μm) from the same individual with a fully binucleate tapetum (arrows; although some cells appear uninucleate, they are truly binucleate, with the second nucleus being out of the optical plane). (C-E) Tapetum binucleation exhibits clustering. In this experiment, three anthers that had about half of their tapetum cells binucleate (average ∼53% binucleate) were analyzed. For each binucleate and uninucleate cell, neighbors were categorized as binucleate or uninucleate (total: 87 cells) (C) Binucleate cells are more likely to have binucleate neighbors and (D) uninucleate cells are more likely to have uninucleate neighbors. (E) A representative anther from C and D. Tapetum binucleation exhibits clustering (pink, uninucleate cells; green, binucleate cells). (F, G) Anthers from a 48 hr EdU in planta incorporation (12 injections, 4 hours apart) displayed clustering. Numbers indicate the number of cells circled in a given “clone”. [Solid circles, brightest cells (recent and/or repeated EdU incorporation); dashed circles, cells of medium brightness (one or more divisions away from most recent EdU incorporation); dotted circles, cells with no EdU staining visible (dropouts or slowly cycling).] (H-J) Middle layer and tapetum cells exhibit clustering. (H) Groups of two or four middle layer cells are outlined in an 1125 μm anther. (I) In the same anther, the same outlines perfectly overlay groups of four or eight tapetum cells. From this image, it appears that new cell walls build during anticlinal divisions of the middle layer and tapetum are continuous, and that the tapetum at this stage is exactly one round of division ahead of the middle layer. We hypothesize that these groups of 2, 4, or 8 middle layer and tapetum cells all share a common, single spl precursor, and that each spl cell functions as a developmental unit. (J) Model of middle layer and tapetum clustering showing the overlap of H and I. Scale bars: (A,B) = 10 μm; (E-I) = 25 μm.