Plant cell division

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.

Reactive oxygen species (ROS) are undesirable byproducts of the aerobic life.¹ The most common between them are: the singlet oxygen, the superoxide anion, the hydroxyl radicals and the hydrogen peroxide. They are produced in several cell compartments, including mitochondria, chloroplasts, peroxisomes, as well as in the apoplast.^2,3 Plant cells have developed astonishing abilities not only to manage the internal and external sources of ROS via enzymatic and non-enzymatic scavenging mechanisms, but also to use free radicals as signaling molecules.⁴ There is convincing evidence that ROS function as key regulators of plant development. They are implicated in a variety of biological processes such as root-hair and pollen tube growth, hormonal responses and biotic and abiotic stress responses.^5-7 Moreover, ROS trigger signal transduction pathways and, even more, control gene expression.²

In animal cells, ROS are involved in cell proliferation regulating transition through specific cell cycle checkpoints.⁸ Similarly, plant cell cycle progression depends on redox sensing. ROS are able to modulate the activity of the cyclin dependent kinases.^9,10 Moreover, apart from acting to protect plant cells from oxidative damage, antioxidants are also implicated in cell cycle control influencing specific cell cycle transitions. During different stages of the cell cycle, the levels of glutathione and ascorbate seem to fluctuate¹¹ along with ROS levels.^12,13

Despite the extensive information regarding ROS signaling in cell cycle regulation, the available knowledge on the relationship between ROS and plant cell mitosis and cytokinesis (cell division) is very limited. Plant cell division is performed by specific microtubule (MT) arrays characterized by extensive and continuous reorganization. Recent evidence revealed that ROS homeostasis is critical for plant cell division.¹⁴ In this article, the available knowledge on ROS implication in plant cell division and, particularly, in MT organization is reviewed. Moreover, the roles of proteins, which are key components of ROS signaling processes during plant cell division, are discussed.

ROS Homeostasis and Plant Cell Division

Experimental disturbance of ROS homeostasis in root-tips of Triticum turgidum and Arabidopsis thaliana deeply affects mitosis¹⁴ and cytokinesis. ROS imbalance caused significant delay in the transition from prophase to prometaphase, interfered with nuclear envelope dynamics, affected prometaphase and anaphase chromosome movement and delayed cell exit from telophase. Cytokinesis was also greatly affected. As a result, multinucleate or polyploid cells were formed. Notably, mitotic and cytokinetic aberrations were found by using menadione, a quinone that induces elevated ROS levels. The aberrations were very similar to those detected after treatment with diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, and N-acetyl cysteine, a ROS scavenger, which reduces ROS levels. These findings are strongly supported by data derived from the study of dividing root-tip cells of the rhd2 A. thaliana mutants, lacking the function of the RHD2/AtRBOHC protein that is differentially expressed in roots.^15,16 The rhd2 plants exhibit short root hairs as well as shorter roots than the wild type.¹⁵ Definite rhd2 root cell types displayed aberrations comparable to those found after treatment with DPI.¹⁴ Some of them seem to be the outcome of misorganization and malfunction of the tubulin cytoskeleton involved in cell division.

Nuclear envelope dynamics

ROS interference with nuclear envelope dynamics was evidenced by the delayed breakdown of the nuclear envelope at late prophase and its delayed reconstitution at telophase.¹⁴ The affected cells were accumulated at the end of prophase, while their transition to prometaphase was highly delayed or even prevented. It is well known that cyclin dependent kinases are involved in phosphorylation of the lamin network, thus triggering nuclear envelope breakdown.¹⁷ Relying on the above and on ROS interference with the activity of cyclin dependent kinases,⁹ the nuclear envelope persistence in late prophase plant cells affected by ROS modulators, can be correlated with lamin malfunction induced by ROS imbalance. Recently, it has been also found that in animal cells oxidative stress induces lamin accumulation.¹⁸

Alternatively, it must be considered that ROS homeostasis interferes with Ca²⁺ homeostasis¹⁵ and that nuclear envelope disintegration and reconstitution require definite local Ca²⁺ oscillations.¹⁹ Therefore, it might be suggested that the aberrant nuclear envelope behavior is caused by the imbalance of cytoplasmic Ca²⁺ homeostasis that is induced by loss of ROS homeostasis. ROS can activate Ca²⁺ channels in roots,¹⁵ while oxidative stress is accompanied by Ca²⁺ release in cytosol.²⁰

Organization of tubulin cytoskeleton

ROS imbalance causes multiple effects on tubulin cytoskeleton in dividing root-tip cells,¹⁴ which are structurally expressed as follows: (1) Preprophase band (PPB) formation was prevented in many preprophase/prophase cells, while when it was present it was appeared highly aberrant, consisting of atypical tubulin polymers. (2) The perinuclear tubulin polymer formation in prophase cells was inhibited. In case of their presence, they failed to assemble a bipolar prophase spindle. (3) The metaphase and anaphase spindle organization was also perturbed. These mitotic spindles displayed tubulin strands randomly oriented incapable to form bipolar systems. (4) The phragmoplast, made of atypical tubulin polymers, was aberrant, and its expansion toward cell cortex was delayed (Fig. 1C; cf. Fig. 1A). Consequently, in many affected cytokinetic cells the cell plate was absent or highly atypical (Fig. 1D; cf. Fig. 1B). Abnormalities in spindle and phragmoplast organization may be attributed to the replacement of MTs by atypical tubulin polymers.¹⁴ A more detailed discussion on atypical tubulin polymer formation upon ROS imbalance is presented below.

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.

Concerning the absence of perinuclear tubulin polymers in prophase cells, it might be suggested that the perinuclear MT-organizing center activation is affected by the presence of ROS modulators. AtTPX2 is a protein, which seems to be implicated in the perinuclear MT-organizing center activation. It is believed that, after its transport from nucleus into cytoplasm, this protein is phosphorylated by aurora kinases.²¹ In animal cells, ROS are implicated in the activation of the α-aurora kinase. The phosphorylated form of this kinase is absent from the centrosome of DPI treated animal cells.^22,23 Then, it may be extrapolated that cells affected by ROS modulators aurora kinases fail in phosphorylating the AtTPX2 protein. In A. thaliana, α-aurora kinases have been also implicated in the determination of the cell division plane.²⁴ Therefore, ROS imbalance, modifying the activity of aurora kinases, might prevent PPB formation in many affected cells.

Chromosome movement

ROS modulators interfere with the metaphase chromosome alignment on the equatorial spindle plane as well as with the anaphase chromosome movement.¹⁴ This may be due to the inability of the affected cells to assemble functional spindles and to inhibition of chromatid separation. Besides, the latter process depends on the action of proteases named separases. Prior to chromatid segregation, separases are bound to a small protein, the securin that inhibits their action. The anaphase promoting complex is responsible for the liberation of separases, which cleave the cohesin complex holding daughter chromatids together.^25,26 The function of separases is well known in dividing animal cells, whereas recent data indicate their participation in plant cell division.^26,27 In animal cells, ROS overproduction prevents the anaphase chromosome separation. Treatment with hydrogen peroxide inhibits anaphase promoting complex and subsequently blocks the separase-securin disassociation, delaying cell cycle progress.²⁵ Moreover, in animal as well as in plant cells aurora kinase is implicated in chromosome segregation.²⁸ At least in animal cells experiencing low ROS levels, α-aurora kinase expression is affected.^22,23 Besides, the assembly of non-functional mitotic spindles and phragmoplasts can be explained by the presence of atypical tubulin polymers, which is induced by the loss of ROS homeostasis.¹⁴ Macrotubules (tubulin tubules exhibiting diameter greater than that of MTs) form in low ROS levels and tubulin paracrystals in elevated ones (see below).

Imbalanced ROS Levels and Atypical Tubulin Polymer Formation

In animal cells, the loss of ROS homeostasis affects MT cytoskeleton. Treatment of Rat1 fibroblasts with DPI, which leads to low ROS production, caused disassembly of tyrosinated MTs and their replacement by detyrosinated ones.²⁹ Besides, oxidative stress applied on human cortical neuron cell lines, induced MT loss, which was followed by MT rearrangement.³⁰

In plants, the first information on the functional relationship between ROS and MT cytoskeleton was derived from the study of cellular responses to biotic or abiotic stress. Salinity stress followed by ROS overproduction³¹ led to MT depolymerization and subsequently to MT reorganization, which is probably required in order for cells to withstand salt stress.³² Furthermore, Verticillum dahliae toxins applied on A. thaliana leaves induced a rapid increase of ROS levels, which in turn resulted in MT disruption.³³ Interestingly, treatment of A. thaliana leaves with DPI that reduces ROS levels alleviates the effects of V. dahliae toxins on the MT cytoskeleton. In addition, Nick³⁴ noted that biotic stress perception depends on MTs and is also mediated by RBOHC (respiratory burst oxidase homolog), the plant homolog of the NADPH oxidase that upon activation catalyzes ROS production in the apoplast.

In dividing plant cells, the serious effects of the experimentally induced ROS homeostasis disruption on MT cytoskeleton were monitored in root-tip cells of the angiosperms T. turgidum and A. thaliana.¹⁴ Under either low or increased ROS levels, MTs coexisted with atypical tubulin polymers. In DPI and N-acetyl cysteine treated cells, the majority of tubulin polymers were macrotubules, while in menadione treated cells, tubulin cytoskeleton was mainly consisted of tubulin paracrystals.¹⁴ Unlikely, the oxidative stress induced by hydrogen peroxide mimics the effects of DPI. In root-tip cells of T. turgidum, treatment with hydrogen peroxide resulted in replacement of MTs by tubulin strands (Fig. 2A; cf. Fig. 2B) similar to those observed in DPI treated cells (Fig. 2B; cf. Fig. 2C). This may be attributed to the mode by which hydrogen peroxide exerts its function. Contrary to other oxidative stress generators,³⁵ the exogenously applied hydrogen peroxide mainly accumulates in the apoplast and diffuses into cytoplasm through the plasma membrane.³⁶

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.

Western blotting analysis showed that loss of ROS homeostasis disturbs the levels of α-tubulin in roots of T. turgidum. Treatment with DPI, N-acetyl cysteine and hydrogen peroxide caused increase of α-tubulin levels, while on the contrary menadione treatment resulted in reduced α-tubulin levels (Fig. 3). This implies a direct effect of increased free radicals on α-tubulin (see also ref. ³⁰). Similarly, the oxidative stress generated by quinones in human brain cell extracts reduced significantly the levels of β-tubulin.³⁷

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).

Moreover, immunoblotting revealed that treatment of roots with ROS modulators induces α-tubulin acetylation as well as a slight decrease of the tyrosinated α-tubulin levels (Fig. 3). Since acetylated tubulin is absent from the control roots (Fig. 3), it might be suggested that macrotubules and tubulin paracrystals consist of or contain acetylated tubulin. These data strengthen previous ones derived by indirect immunofluorescence, which showed the presence of acetylated tubulin in the atypical tubulin polymers.¹⁴ During oxidative stress, tubulin acetylation may represent a cell response, necessary to protect tubulin from the elevated ROS levels.

As far as MT disappearance associated with the disturbance of ROS homeostasis is concerned, several hypotheses can be formulated. Thus, the increased ROS levels may lead to direct MT disruption through: (A) elevation of Ca²⁺ levels,^14,32 (B) oxidative modifications of tubulin,³⁷ and/or (C) inactivation of MT-associated proteins (MAPs).³³ However, the possibility that low ROS levels induce MT disintegration by interference with MT dynamic instability cannot be also excluded.¹⁴ It has been supported that DPI destructs the unstable MTs of Rat1 fibroblasts by uncoupling their minus end from centrosome, thus triggering MT disintegration.²⁹

Signaling Mechanisms Inducing ROS Production: The Crucial Role of the NADPH Oxidase

Ca²⁺ levels have been implicated in the activation of NADPH oxidases,¹⁶ enzymes playing a key role in ROS signaling. They are considered as enzymes involved in “deliberating” ROS production.³⁸ These membrane-bound enzymes transfer electrons from NADPH to molecular oxygen generating superoxide anions in the apoplast. Superoxide anions could react with H₂O to produce hydrogen peroxide.³⁹ Several indications suggest that ROS production catalyzed by NADPH oxidase influences MT organization. Some of the protein molecules involved in regulation of the NADPH oxidases have been also implicated in MT organization. Among others, proteins that activate NADPH oxidases are phospholipase D (PLD), phosphatidylinositol 3-kinase (PI3K) and ROP-GTPases. A similar model describing the control of the actin cytoskeleton in root-hair tip growth has already been proposed by Samaj et al.⁴⁰ The role of these proteins during cell division is discussed below.

Direct evidence, which favors the idea that ROS production by NADPH oxidases is important for the organization of tubulin cytoskeleton and cell division, has been derived from the study of dividing root-tip cells of the rhd2-5 and rhd2-6 A. thaliana mutants.¹⁴ They lack the RBOHC, one of the ten A. thaliana NADPH oxidases, which is differentially expressed in roots.¹⁶ Specific root cell types of these mutants, i.e., root-hairs, rhizodermal and endodermal cells, exhibited arrays of tubulin polymers consisted of macrotubules and MTs. The percentage of macrotubules varied among different root cell types,¹⁴ but the pattern of their appearance was in accordance with the pattern of expression of the RHD2 protein in control A. thaliana roots.¹⁵ The tubulin cytoskeleton defects displayed by these mutants can be safely attributed to the lack of the NADPH oxidase RBOHC and explain the mitotic¹⁴ and cytokinetic disorders (Fig. 4D; cf. Fig. 4A) found in many cells. This strengthens findings obtained after treatment of root-tip cells with DPI and shows that the disturbance of the tubulin cytoskeleton is due to the reduction of ROS production.

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.

Phosholipase D

PLDs are membrane phosphodiesterases involved in ROS responses. They also trigger ROS production.⁴¹ In guard cells, PLDα1 generates phosphatidic acid (PA) in response to abscisic acid and contributes to ROS mediated stomatal closure.⁴² PA activates the main NADPH oxidases of guard cells, RBOHD and RBOHF. Moreover, PA binds directly to RBOHs resulting in their activation.^38,42 Besides, a significant amount of experimental work shows that, in plant cells, PA levels affect MT organization. For example, 1-butanol, which functions as substrate of PLD and results in inhibition of PLD derived PA production, disrupts MT arrays and blocks cell division in Silvetia compressa zygotes.⁴³ In addition, 1-butanol induces extensive MT depolymerization in BY-2 cells and A. thaliana seedlings^44,45 and affects tubulin cytoskeleton in plasmolyzed root-tip cells of T. turgidum.⁴⁶ Therefore, it is tempting to suggest that PLD, among others, influences tubulin cytoskeleton, via the NADPH oxidase dependent ROS generation.

Phosphatidylinositol 3-kinase

Phosphatidylinositol 3-phosphate is a phosphoinositide generated by PI3K, which is involved in ROS production.⁴⁷ In animal cells, phosphatidylinositol 3-phosphate seems to stimulate ROS production by direct binding to the PX domain of the NADPH oxidase.⁴⁸ In plants, this enzyme is required for root-hair growth. PI3K inhibition by its specific inhibitor LY294002 prevented tip growth in root-hairs and simultaneously reduced ROS production.⁴⁹ However, RBOHC is required exclusively for ROS generation at the onset of root-hair elongation. Other ROS generators seem to be implicated in PI3K induced ROS production at the late stages of root-hair elongation.⁴⁹

Treatment of T. turgidum and A. thaliana root-tips with LY294002 disturbed the organization of tubulin cytoskeleton (Fig. 4E and F; cf. Fig. 4B and C) and seriously affected cell division. The effects were very similar to those recorded after DPI treatment. These findings allow the hypothesis that ROS generation, which is stimulated by the action of PI3K in dividing cells, participates in ROS homeostasis mechanisms. However, at present we are not in position to estimate RBOHC contribution in ROS production driven by PI3K.

Rho-related proteins

In plants, the ROP-GTPases seem to function as molecular rheostats in sensing oxygen deprivation.⁵⁰ These proteins are involved in RBOH activation. For example, in rice NADPH oxidase activation is mediated by binding to Rac GTPase.⁵¹ On the other hand, kinesins and MAPs are considered as effector molecules of Rho-related GTPases, thus regulating MT organization and dynamics.^52,53 However, it remains unclear whether cytoskeleton signaling via ROP-GTPases includes ROS signaling (see also ref. ⁵²).

ROS Signaling Transduction, Organization of Tubulin Cytoskeleton and Cell Division

MAPs

Recent data suggest that MAP65 is involved in the formation of atypical tubulin polymers induced by ROS modulators.¹⁴ Immunolocalization of MAP65-1 proteins in A. thaliana root-tips revealed the presence of MAP65-1 on macrotubules and in tubulin paracrystals. Moreover, ΜΑP65 proteins participate in the assembly of tubulin paracrystals formed after colchicine treatment.⁵⁴ MAP65 is activated after phosphorylation by MPK4 and MPK6. MPK4 and MPK6 are key components in ROS signaling transduction.⁵⁵ Therefore, it might be suggested that MAP65 activation is regulated, at least in part by ROS levels, to promote MT-bundling in normal conditions and the atypical tubulin polymer formation under disturbed ROS levels. Consequently, this protein seems to respond to changes of MAPK signaling induced by ROS levels.

MAPKs

MPK4 is a MAPK involved in abiotic signal perception and its action as ROS responsive MAPK has been well appreciated.⁵⁵ In addition, MPK4 co-localized with MT arrays in A. thaliana root cells in a pattern similar to that of MAP65-1.⁵⁶ MPK4 plays a role in the transition from mitosis to cytokinesis, whereas the mpk4 mutant exhibits aberrant spindles and phragmoplasts.⁵⁷ Therefore, MPK4, apart from the abiotic signal transduction, appears to be implicated in sensing alterations in ROS levels as well as in the regulation of MT organization during plant cell division. MPK4 activation depends on its phosphorylation by MEKK1. This MAPKKK is involved in the expression of numerous genes implicated in cellular redox control.⁵⁸ Relation between MEKK1 and MPK4 dependent MT regulation has been recently described.⁵⁹

In animal cells, the p38 MAPK is a stress responsive MAPK, which is upregulated under oxidative stress and downregulated when ROS levels decrease.^60,61 A p46 MAPK, displaying immunological and pharmacological properties similar to those of the p38 MAPK, is activated in plants experiencing osmotic stress. Its phosphorylation seems to induce changes in tubulin cytoskeleton under hyperosmotic conditions, stimulating macrotubule formation.⁶² Considering that under disturbed ROS levels the root-tip cells assemble atypical tubulin polymers,¹⁴ it was investigated whether the p38-like kinase (p46 MAPK) is involved in the formation of these tubulin polymers.

In this direction, the specific p38 MAPK inhibitor SB203580 was applied together with drugs creating oxidative stress on T. turgidum roots. In presence of the inhibitor, the intensity of the atypical tubulin polymer appearance was alleviated (Fig. 4H; cf. Fig. 4G). This is in accordance with the hypothesis that p38 like MAPK is activated in plants experiencing oxidative stress and mediates the formation of atypical tubulin polymers, probably to prevent tubulin damage by oxidative modification.¹⁴ Moreover, application of the SB203580 together with DPI or N-acetyl cysteine also seemed to alleviate atypical tubulin polymer assembly in root-tip cells (Fig. 4J; cf. Fig. 4I). Therefore, in cells treated with ROS modulators in the presence of the p38 MAPK inhibitor, the organization of the tubulin cytoskeleton is comparable to that of the untreated cells (Figs. 4H and J; cf. Fig. 2A). This was not expected since p38 kinase, at least in animal cells, is activated under oxidative stress and is downregulated under low ROS levels.^60,61

Reactive Nitrogen Species

Emerging evidence reveals that apart from ROS, the reactive nitrogen species (RNS) play a particular role in cell cycle regulation.^63,64 In several cases, ROS act cooperatively with RNS to stimulate responses through the same signaling cascades.⁶⁵ They can also co-interact, e.g., nitric oxide anion can interact with superoxide anion to form peroxynitrite.⁶⁶ Nitric oxide is produced by non enzymatic mechanisms in the apoplast or inside the cells by the activation of nitric oxide synthase putative enzymes.⁶⁷ RNS are produced simultaneously with ROS in biotic or abiotic stress conditions. The role of RNS in MT cytoskeleton has been recently reviewed.⁶⁸ As in the case of ROS, nitric oxide also induces tubulin post transcriptional modifications (tyrosine nitration). Nitric oxide donors and scavengers regulate MT organization. In particular, RNS homeostasis seems to be critical for the proper cortical MT formation. Therefore, studies concerning ROS, MT organization and cell division should also consider RNS involvement in the future.

Conclusion and Future Perspectives

ROS signaling triggered “a new wave” of research in plant cells.⁴ Among others, information accumulated so far allow the hypothesis that ROS are molecules functioning as signaling components during cell division, through complex signaling pathways. The fluctuations of oxidants and antioxidants during cell division^11-13 support ROS implication in this process, while ROS imbalance induces formation of atypical tubulin polymers.¹⁴

ROS production seems to be induced by environmental, hormonal or other intercellular signals. NADPH oxidase releases ROS in the apoplast that enter into the cytoplasm. ROS production by NADPH oxidases is triggered by PLD, PI3K or ROP-GTPases. Intracellular ROS level oscillations keep pace with respective antioxidant oscillations. Subsequently, ROS acting as signaling molecules contribute to the establishment of Ca²⁺ gradients and participate in the control of regulatory proteins such as cyclin dependent kinases, MAPs and possibly aurora kinases. Thus, ROS are implicated in the regulation of cell cycle progress, organization of MT arrays, nuclear envelope dynamics and cell plate formation (Fig. 5).

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

However, further work is needed to elucidate what exactly ROS do and how. Particular attention should also be paid to identify the role of each type of ROS in cell division, the sites of their production, as well as their targets. In this direction, examination of MT organization in mutants lacking proteins involved in ROS production and scavenging would also improve the existing knowledge on the relation between ROS and plant cell division. Moreover, further transcriptome and proteome analysis under conditions creating low or elevated ROS levels would reveal missing links in the effects of ROS on MTs, whereas analysis using oxyblot technique could show oxidative modifications in several proteins induced by ROS. In any case, experiments with ROS modulators should be carefully interpreted, since ROS imbalance may cause cell damage.

Glossary

Abbreviations:

DPI

diphenylene iodonium

MAP

microtubule associated protein

MT

microtubule

PA

phosphatidic acid

PI3K

phosphatidylinositol 3-kinase

PLD

phospholipase D

PPB

preprophase band

ROS

reactive oxygen species

RBOHC

respiratory burst oxidase homolog C

RNS

reactive nitrogen species

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.