Anther Morphological Development and Stage Determination in Triticum aestivum

Abstract

Anther development progresses through 15 distinct developmental stages in wheat, and accurate determination of anther developmental stages is essential in anther and pollen studies. A detailed outline of the development of the wheat anther through its entire developmental program, including the 15 distinct morphological stages, is presented. In bread wheat (Triticum aestivum), anther developmental stages were correlated with five measurements, namely auricle distance, spike length, spikelet length, anther length and anther width. Spike length and auricle distance were shown to be suitable for rapid anther staging within cultivars. Anther length is an accurate measurement in determining anther stages and may be applicable for use between cultivars. Tapetal Programmed Cell Death (PCD) in wheat begins between late tetrad stage (stage 8) and the early young microspore stage (stage 9) of anther development. Tapetal PCD continues until the vacuolate pollen stage (stage 11), at which point the tapetum fully degrades. The timing of tapetal PCD initiation is slightly delayed compared to that in rice, but is two stages earlier than in the model dicot Arabidopsis. The MYB80 gene, which encodes a transcription factor regulating the timing of tapetal PCD, reaches its peak expression at the onset of tapetal PCD in wheat.

Keywords: MYB80; Triticum aestivum; anther; apoptosis-like PCD; developmental staging; tapetum.

Figure 1

Plant measurements utilized in predicting anther developmental stage in wheat. (A) Auricle distance (AD) identified on a wheat tiller. (B) Spike length (SL) indicated on a wheat spike with a central spikelet indicated with an asterisk. (C) Spikelet length (SpL) indicated on a spikelet. (D) Anther length (AL) and anther width (AW) identified against an anther.

Figure 2

Transverse sections of wheat anthers from developmental stages 1–15. Samples were obtained from Halberd and Cranbrook cultivar wheat plants. Samples were fixed with LR White resin and stained using 1% toluidine blue. L1, 1st Cell layer; L2, 2nd Cell Layer; L3, 3rd Cell layer; Pa, Parietal Tissue; Sp, Sporogenous Tissue; E, Epidermis; En, Endothecium; ML, Middle Layer; T, Tapetum; L, Lacunae; StR, Stomium Region; MMC, Microspore Mother Cells; Tds, Tetrads; YM, Young Microspores; VM, Vacuolate Microspores; VP, Vacuolate Pollen; PG, Pollen Grains; MC, Meiotic Cells; V, Vascular Region; C, Connective Tissue; CC, Central Callose. Scale bar is 100 μm for stages 1–7 and 200 μm for stages 8–15.

Figure 3

Anther developmental stages compared with five plant measurements from four wheat cultivars. Anther stages compared to auricle distance (A), anther length (B), anther width (C), spike length (D), and spikelet length (E). Anthers were fixed and transverse sectioned before being staged using brightfield microscopy. For each stage, averages are shown with error bars representing standard error. Auricle distance measurements are shown for Halberd (red), Cranbrook (blue), Young (green) and Wyalkatchem (purple). Stages are numbered based on descriptions given in Table 1. Linear regression lines are shown for each cultivar in each graph.

Figure 4

TUNEL assay of wheat cv. Halberd anthers from stage 7 to 12. Confocal microscopy of TUNEL assay in 6 μm sections of Halberd cultivar wheat anthers. Red signal is propidium iodide counter stain. Green identifies fluorescein tagged nuclei undergoing apoptosis-like Programmed Cell Death. White arrows indicate tapetal cells undergoing apoptosis-like PCD. Yellow arrows indicate tapeta where no TUNEL signal is observable. T, Tapetum; TR, Tapetal Remnant; MC, Meiotic Cell; Tet, Tetrad; YM, Young Microspore; VM, Vacuolate Microspore; VP, Vacuolate Pollen; PG, Pollen Grain. Numbers indicate anther developmental stage. Scale bars = 75 μm.

Figure 5

MYB80 expression in the anthers of Halberd cultivar wheat across a variety of anther developmental stages. Each stage is the average expression of four biological replicates, each with 4 technical replicates. Expression is shown as relative transcript abundance, calculated as 2^−ΔCt. Transcript levels are calculated relative to the average of the expression of ADP-ribosylation factor (ARF) (Ta2291) and Cell Division Control protein (CDC) (Ta54227). Error bars represent standard error.

Figure 6

Normalized count values for putative wheat orthologs of six rice genes involved in tapetal PCD regulatory processes. Each rice gene has three wheat orthologs, or six in the case of TDR. The genes are identified by their rice ortholog name and which wheat genome they are present on (A, B, or D). Counts from RNAseq experiment performed on wheat cv. Halberd anther tissue, collected at meiosis/tetrad stages (blue) or tetrad/young microspore stages (red) as measured with auricle distance. Each bar represents the average of three replicates and error bars represent standard error.