Tubulin Acetylation Mediates Bisphenol A Effects on the Microtubule Arrays of Allium cepa and Triticum turgidum

Abstract

The effects of bisphenol A (BPA), a prevalent endocrine disruptor, on both interphase and mitotic microtubule array organization was examined by immunofluorescence microscopy in meristematic root cells of Triticum turgidum (durum wheat) and Allium cepa (onion). In interphase cells of A. cepa, BPA treatment resulted in substitution of cortical microtubules by annular/spiral tubulin structures, while in T. turgidum BPA induced cortical microtubule fragmentation. Immunolocalization of acetylated α-tubulin revealed that cortical microtubules of T. turgidum were highly acetylated, unlike those of A. cepa. In addition, elevation of tubulin acetylation by trichostatin A in A. cepa resulted in microtubule disruption similar to that observed in T. turgidum. BPA also disrupted all mitotic microtubule arrays in both species. It is also worth noting that mitotic microtubule arrays were acetylated in both plants. As assessed by BPA removal, its effects are reversible. Furthermore, taxol-stabilized microtubules were resistant to BPA, while recovery from oryzalin treatment in BPA solution resulted in the formation of ring-like tubulin conformations. Overall, these findings indicate the following: (1) BPA affects plant mitosis/cytokinesis by disrupting microtubule organization. (2) Microtubule disassembly probably results from impairment of free tubulin subunit polymerization. (3) The differences in cortical microtubule responses to BPA among the species studied are correlated to the degree of tubulin acetylation.

Keywords: bisphenol A; cytokinesis; microtubules; mitosis; tubulin acetylation.

Figure 1

Tubulin immunolocalization in interphase root cells of the two plant species studied, either untreated or bisphenol A (BPA)-treated (as depicted), at a single cortical confocal laser scanning microscope (CLSM) section (A, B, E, G) or a maximum projection of serial sections (C, D, F, H). The plant species and treatment regime are similarly noted in all the following figures. Scale bar: 10 μm.

Figure 2

(A,C,E,G) Tubulin immunolocalization (green, projections of CLSM sections) and (B,D,F,H) propidium iodide DNA staining (red, single CLSM sections) in pre-prophase root cells. Arrows point to aberrantly organized pre-prophase microtubule bands of BPA-affected cells. Note the absence of perinuclear microtubules in (F) and (H) in contrast with the control (arrowheads in (B,D)). Scale bar: 10 μm.

Figure 3

(A,C,E,G) Tubulin immunolocalization (green, projections of CLSM sections) and (B,D,F,H) propidium iodide DNA staining (red, single CLSM sections) in prophase (see chromatin condensation) root cells. Arrows in (E,G) point to aberrantly organized pre-prophase microtubule bands of BPA-affected cells. Note the absence of perinuclear spindles in (F,H). Scale bar: 10 μm.

Figure 4

(A,C,E,G,I,K) Tubulin immunolocalization (green) and (B,D,F,H,J,L) propidium iodide DNA staining (red) in untreated metaphase (A–D) and anaphase (I–L) cells, as well as BPA-affected mitotic I–L) root cells. In BPA-treated cells the metaphase or anaphase status could not be distinguished. All images are projections of CLSM sections. Scale bar: 10 μm.

Figure 5

(A–P) Tubulin immunolocalization (green) and (B,D,F,H,J,L,N,P) propidium iodide DNA staining (red) in telophase/cytokinetic root cells. Arrowhead in (F) points to a lagging chromosome arm, while arrowhead in (K) points to unexpanded phragmoplast. Note the extensive presence of branched phragmoplast conformations (E–J,M,N). (A,C,E,G,H,I,K,M,O) Maximum projections of CLSM sections; (B,D,F,J,L,N,P) single sections. Scale bar: 10 μm.

Figure 6

Maximum projections of CLSM sections, after tubulin immunolocalization in interphase; T. turgidum root cells recovering after oryzalin treatment, either in (A) water or in (B) BPA solution. Scale bar: 10 μm.

Figure 7

Maximum projections of CLSM sections, after tubulin immunolocalization in interphase root cells treated with taxol and/or BPA (as noted on images A–F). Scale bar: 10 μm.

Figure 8

(A–H) Acetylated α-tubulin immunolocalization (green) and (B–H) propidium iodide DNA staining (red) in untreated root cells. The inset in (E) depicts the interphase nucleus of the cell. All images are single CLSM sections. Scale bar: 10 μm.

Figure 9

Western blot depicting the content of acetylated and total α-tubulin in roots. GAPDH is used as a loading control. TSA: trichostatin A.

Figure 10

Maximum projections of CLSM sections, after acetylated α-tubulin (A,B) or total α-tubulin (C,D) immunolocalization in control and variously-treated (as noted) A. cepa interphase root cells. Scale bar: 10 μm.

Figure 11

Tubulin immunolocalization (green, A, B1, C, D1, E1, F, G1, H) and propidium iodide DNA staining (red, B2, D2, E2, F, G2, H) in root cells recovering after BPA treatment for duration noted on the figures. Arrows in (F) point to an atypical phragmoplast, while arrows in (G2) point to cell plate remnant in a post-cytokinetic cell. Images (D1, F, H) are maximum projections of CLSM sections, while the rest of the images are single CLSM sections. Scale bars: 10 μm.

Figure 12

Tubulin immunolocalization (green, A1, B1, C–E, F1, G1, H, I, J1, K1, L1, M1, N1, O1, P1), propidium iodide DNA staining (red, A2, B2, C–E, F2, G2, H, I2) and DAPI DNA staining (blue, J2, K2, L2, M2, N2, O2, P2) in root cells recovering after BPA treatment. Arrows in (C–E) point to pre-prophase band profiles, arrows in (I2) point to cell plate remnant in post-cytokinetic cell, arrows in (J1) and (K1) point to interphase microtubules, arrow in (L1) points to a metaphase mini-pole, arrow in (M2) points to chromosome arms diverging laterally, while arrows in (P1) point to pre-prophase band profiles. Images (A, B, F, H2) depict maximum projections of CLSM sections, images (C–E, G, H1, I) are single CLSM sections, while images (J–P) are conventional fluorescence micrographs. Scale bars: 10 μm.