Plant cell division: ROS homeostasis is required

Abstract

Accumulated evidence indicates that ROS fluctuations play a critical role in cell division. Dividing plant cells rapidly respond to them. Experimental disturbance of ROS homeostasis affects: tubulin polymerization; PPB, mitotic spindle and phragmoplast assembly; nuclear envelope dynamics; chromosome separation and movement; cell plate formation. Dividing cells mainly accumulate at prophase and delay in passing through the successive cell division stages. Notably, many dividing root cells of the rhd2 Arabidopsis thaliana mutants, lacking the RHD2/AtRBOHC protein function, displayed aberrations, comparable to those induced by low ROS levels. Some protein molecules, playing key roles in signal transduction networks inducing ROS production, participate in cell division. NADPH oxidases and their regulators PLD, PI3K and ROP-GTPases, are involved in MT polymerization and organization. Cellular ROS oscillations function as messages rapidly transmitted through MAPK pathways inducing MAP activation, thus affecting MT dynamics and organization. RNS implication in cell division is also considered.

Figure 1. Cytokinetic control (A and B) and treated with ROS modulators (C and D) T. turgidum root-tip cells as they appear after tubulin immunolabeling (A and C) and DIC optics (B and D). The asterisks mark the daughter nuclei and the arrows point to cell plates. Aberrant phragmoplast expansion follows treatment with N-acetyl cysteine (C; cf. A), while a dilated cell plate (D; cf. B) forms in the presence of diphenylene iodonium. Treatments: (C) NAC 250 μM, 1 h; (D) DPI 25 μM, 1 h. Bar: 10 μm.

Figure 2. Control (A) and treated with hydrogen peroxide (B) and diphenylene iodonium (DPI) (C) T. turgidum root-tip cells as they appear after tubulin immunolabeling. The effects of hydrogen peroxide treatment on tubulin cytoskeleton (B; cf. A) are comparable to those induced by DPI (C; cf. B). Treatments: (B) H₂O₂ 4 mM, 1 h; (C) DPI 25 μM, 1 h. Bar: 10 μm.

Figure 3. Western blot analysis of α-tubulin, acetylated and tyrosinated α-tubulin levels after treatment of T. turgidum roots with ROS modulators for 1 h. Twenty micrograms of total protein extract were loaded per well in every case. Protein amount was assessed with Bradford reagent (CONT, dH₂O; DPI, diphenylene iodonium 25 μM; ΝΑC, N-acetyl cysteine 250 μM; MEN, menadione 25 μM; H₂O₂ 4mM).

Figure 4.A. thaliana (A and D) and T. turgidum (B, C and E--J) root-tip cells as they appear after tubulin immunolocalization. (A and D) Cytokinetic cells from wild type (A) and rhd2-6 (D) A. thaliana roots. Note the differences in phragmoplast organization. (B, C, E and F) Prometaphase (B and E) and cytokinetic (C and F) control (B and C) and treated cells with the specific inhibitor of PI3K LY294002 (E and F). The asterisks mark the daughter nuclei. Treatment: LY294002 50 μM, 2 h. (G and I) Atypical tubulin polymers appeared in cells after treatment with ROS modulators (compare Fig. 2A). Treatments: (G) MEN, menadione 25 μM, 1 h; (I) DPI, diphenylene iodonium 25 μM, 1 h. (H and J) The intensity of atypical tubulin polymer formation is clearly alleviated in the presence of the p38 MAPK specific inhibitor SB203580 (compare G and I). Treatments: (H) SB 10 μM, 30 min + SB plus MEN 25 μM, 1 h; (J) SB 10 μM, 30 min + SB plus DPI 25 μM, 1 h. Bar: 10μM.

Figure 5. Simplified diagram describing probable routes of ROS implication in cell division. Abbreviations used: Aurora, Aurora kinases; Cdks, cyclin dependent kinases; MAPs, microtubule associated proteins; MAPK, mitogen activated protein kinase; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase; PI3K, phosphatidylinositol 3-kinase; PLD, phospholipase D; ROP-GTPases, rho-related GTPases of plants; ROS, reactive oxygen species