Abnormal Development of Tapetum and Microspores Induced by Chemical Hybridization Agent SQ-1 in Wheat

<div><p>Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.</p></div

Scanning electron micrograph observations of untreated (A to E) and chemical hybridization agent (CHA)-SQ-1-treated wheat plant (F to J) anthers at the trinucleate stage.

<p>(B, C, G and H) the outer epidermal cells. (D, E, I and J) the inner epidermal cells. Uby indicate the Ubisch body. Scale bars are 1 mm in A and F and are 100 μm in B and G and are 10 μm in C to E and H to J.</p

FDA-stained transverse sections and microspores of untreated and chemical hybridization agent (CHA)-SQ-1-treated wheat plants.

<p>Transverse sections (A to J) and microspores (K to T) treated with fluorescein diacetate (FDA) for the detection of cell viability and membrane integrity of untreated and CHA-SQ-1-treated plants at the tetrad stage (A, F, K and P), early-uninucleate stage (B, G, L and Q), later-uninucleate stage (C, H, M and R), binucleate stage (D, I, N and S) and trinucleate stage (E, J, O and T), respectively, as viewed under a fluorescence microscope (excitation wavelength 450–490 nm). FDA signals appear green in colour, and microspores with relatively weak or no signal were considered to be of low-viability or dead (see white <i>arrowhead</i>). The microspore survival rates are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119557#pone.0119557.s005" target="_blank">S5 Fig</a>. T indicates the tapetum. Scale bars are 50 μm.</p

Development of anthers and microspores in untreated (A to E and K to P) and chemical hybridization agent (CHA)-SQ-1-treated wheat plants (F to J and Q to V).

<p>(A to J) safranin O/fast green-stained transverse sections. (K to O and Q to U) 1% acetocarmine-stained microspores. (P and V) a scanning electron micrograph was used to analyse the mature pollen grains. (A, F, K and Q) the tetrad stage. (B, G, L and R) the early-uninucleate stage. (C, H, M and S) the later-uninucleate stage. (D, I, N and T) the binucleate stage. (E, J, O and U) the trinucleate stage. E, En, ML, T, Tds, Msp and Ap indicate the epidermis, the endothecium, the middle layer, the tapetum, the tetrads, the microspore and the germination aperture, respectively. Scale bars are 50 μm in A to O and Q to U.</p

DNA fragmentation (indicating programmed cell death; PCD) in untreated and chemical hybridization agent (CHA)-SQ-1-treated wheat plants during anther development.

<p>Anther sections in the DNase treatment (Positive control, A to E), untreated (F to J) and CHA-SQ-1-treated plants (K to O) were compared for nuclear DNA fragmentation using the TUNEL assay at the tetrad stage (A, F and K), early-uninucleate stage (B, G and L), later-uninucleate stage (C, H and M), binucleate stage (D, I and N) and trinucleate stage (E, J and O), respectively. Propidium iodide stained nuclei fluoresce red, while green fluorescence is TUNEL-positive nuclei staining. E, En, ML, T, Tds and Msp indicate the epidermis, the endothecium, the middle layer, the tapetum, the tetrads and the microspore, respectively. Scale bars are 50 μm.</p

Comparison of stamens and pistils of untreated (A to E) and chemical hybridization agent (CHA)-SQ-1-treated wheat plants (F to J).

<p>(A and F) the tetrad stage. (B and G) the early-uninucleate stage. (C and H) the later-uninucleate stage. (D and I) the binucleate stage. (E and J) the trinucleate stage. (E and J, top right) the 2% I₂-KI (top right) staining pollen grains. Scale bars are 0.5 mm in A to J and are 50 μm in the top right of E and J.</p