Effects of Inhibitors of Serine/Threonine Protein Kinases on ...

ISSN 1990519X, Cell and Tissue Biology, 2010, Vol. 4, No. 4, pp. 399–409. © Pleiades Publishing, Ltd., 2010. Original Russian Text © Ya.A. Sheremet, A.I. Yemets, K. Vissenberg, J.P. Verbelen, Ya.B. Blume, 2010, published in Tsitologiya, Vol. 52, No. 5, 2010, pp. 389–398.

Effects of Inhibitors of Serine/Threonine Protein Kinases on Arabidopsis thaliana Root Morphology and Microtubule Organization in Its Cells Ya. A. Sheremeta*, A. I. Yemetsa, K. Vissenbergb, J.P. Verbelenb, and Ya. B. Blumea a

Institute of Food Biothechnology and Genomics, National Academy of Sciences of Ukraine, Kiev, Ukraine b University of Antwerp, Antwerp, Belgium *email: yarasheremet@gmail.ñom Received June 9, 2009

Abstract—The effect of different types of serine/threonine protein kinase inhibitors (cyclindependent, Ca2+calmodulin dependent and protein kinase C) on the microtubule organization in cells of Arabidopsis thaliana main primary root zones were investigated in vivo. It was found that the microtubules in epidermal and cortex cells of transition and elongation zones, as well as microtubules in trichoblasts and atrichoblasts of the differentiation zone, were the most sensitive to the action of the investigated protein kinase inhibitors. It was established that, in these types of cells, microtubules change their initial orientation from transverse (oblique) to chaotic or longitudinal relative to the main primary root axis as a result of serine/threonine pro tein kinase inhibition. Microtubules in cells of root meristematic zone, as well as in root hairs, were less sen sitive to the action of tested protein kinase inhibitors. Changes in the orientation of microtubules in cells of primary root zones under the effect of serine/threonine protein kinase inhibitors led to further disturbances in the growth and differentiation processes. It was assumed that the phosphorylation of microtubule proteins, primarily tubulin, could be involved in the regulation of these processes. Key words: microtubules, serine and threonine phosphorylation, protein kinases, inhibitors, Arabidopsis. DOI: 10.1134/S1990519X10040139

Abbreviations used: cAMP, cyclic adenosine mono phosphate; DMSO, dimethylsulfoxide; CDK, cyclin dependent kinase; GFP, green fluorescent protein; MAP, microtubuleassociated protein. INTRODUCTION Earlier we have shown that in plant cells α and β subunits of tubulin undergo posttranslational phos phorylation on both serine (threonine) (Blume et al., 1997, 2008a) and tyrosine residues (Blume et al., 2008b). Using several specific inhibitors of tyrosine kinases and tyrosine phosphatases on the model plant Arabidopsis thaliana expressing the gfpmap4 gene, we have found that phosphorylation on tyrosine residues involved in the organization of microtubules in differ ent types of cells in all growth zones of the primary root (Yemets et al., 2008). Recently, in plant cells, the caseinlike kinase, CKL6, was revealed, which cata lyzes the phosphorylation of serine residues of the tubulin βsubunit at positions 413 and 420 (Ben–Nis san et al., 2008). In another work, it was demonstrated that αtubulin from the tea Camellia sinensis had four sites of phosphorylation by protein kinase C (Fang et al., 2006). Recently, 27 phosphorylated proteins were isolated from the suspension culture of Nicotiana

tabacum; tubulin phosphorylated by the Ca2+calm odulindependent process was also identified among these proteins (Gerber et al., 2006). However, the functional role of the posttranslational phosphoryla tion of microtubules proteins on serine and threonine residues has hardly been investigated. At present, the main types of serine and threonine protein kinases associated with plant microtubules (by analogy with animal cell) have been characterized (Karpov et al., 2009). Hence, it can be suggested that this posttranslational modification plays an important role in the organization of the cortical microtubule network and the regulation of their dynamic properties in plant cells. On the other hand, we have established that both tubulin subunits in plant cells are intensively phospho rylated on serine and threonine residues as a result of the activation of the corresponding protein kinases (cAMPdependent, Ca2+, Ca2+calmodulin), as well as protein kinase C (Blume et al., 2008a). It was found that the use of specific inhibitors of Ca2+calmodulin dependent protein kinases and protein kinase C led to changes in the microtubule organization in root apex cells of 3dayold seedlings of Allium cepa (Blume et al., 2008a). In another work, we showed that the induction of protein hyperphosphorylation by the

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Accretion of length, % 30 Control olomoucine W7 25 H7 staurosporine 20 15 10 5 0

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Fig. 1. Increment of length of A. thaliana primary roots after treatment with inhibitors of protein kinases.

inhibition of serine/threonine protein phosphatases also leads to the disturbance of the microtubule orga nization in plant cells (Sheremet et al., 2009). Thus, to elucidate the role of different types of serine/threonine protein kinases in the phosphoryla tion of proteins of plant cell microtubules and their functional significance, we have attempted to study the effects of inhibitors of several serine/threonine protein kinases on the rearrangement of microtubules in different types of cells. In the present work, we determined the interconnection between inhibition of activities of serine/threonine protein kinases (such as cyclindependent, Ca2+calmodulindependent pro tein kinase and protein kinase C) and the change in the growth parameters and morphology of the primary roots, as well as the microtubule organization in vari ous cells of Arabidopsis thaliana seedling roots. MATERIAL AND METHODS Seedlings of A. thaliana line that expressed the chi meric gene gfpmap4 (Mathur and Chua, 2000) were used in current study. In experiments with inhibitors of serine/threonine protein kinases, we used 4daysold Arabidopsis seedlings obtained as described previously (Sheremet et al., 2009). As an inhibitor of cyclin dependent proteinkinases, we used olomoucine (Sigma, US) dissolved in dimethylsulfoxide (DMSO) at a concentration of 50 mM and stored at –20°C. An inhibitor of Ca2+calmodulindependent protein kinases W7 (Sigma, US) was dissolved in distilled water at +70°C and the stock solution (100 mM) was stored at –20°C. An inhibitor of protein kinase C (Ca2+dependent and stimulated by phospholipid) staurosporine (Sigma, US) was dissolved in DMSO (10 mM) and stored at –20°C. The stock solution (50 mM) of another protein kinase C inhibitor (50 mM) H7 (Sigma, US) was also prepared in DMSO

and stored at –20°C. In experiments, fresh working solutions of inhibitors at various concentrations were prepared in distilled water. The final DMSO concen tration in these solutions did not exceed 0.5% and, as we established previously, is not toxic for plant cells and microtubules (Mitrofanova et al., 2003; Yemets et al., 2005; Ozheredov et al., 2009), which is in agree ment with the results of previous works. In the first series of experiments, we studied the effect of protein kinase inhibitors at various concen trations for 6, 24, and 48 h on the growth and mor phology of roots of 4daysold A. thaliana seedlings. The obtained data were documented using a Canon Power Shot G6 digital camera in the regime of microshooting. The length of the A. thaliana roots was measured by the previously described procedure (Yemets et al., 2008). All experiments were performed in three repeats and the results were processed statisti cally by the commonly accepted methods (Lakin, 1990). To study the shortterm and prolonged effects of specific inhibitors on the organization and orientation of microtubules in cells of various growth zones of the primary root, 4daysold seedlings were treated with the corresponding substances at different concentra tions for 1–48 h. The GFPlabeled microtubules in A. thaliana cells were visualized in vivo by using con focal laser scanning microscope LSM 510 META (Carl Zeiss, Germany). To obtain the threedimen sional image, line 488 of the argon laser was used (excitation at 488–543 nm; emission at 510–540 nm), as well as an immersional objective with a magnifica tion 63× (PlanApochromat). Based on serial optical sections (with the 0.3–0.5 μM interval), 3D models of microtubules were reconstructed using LSM 510 soft ware, version 4.0 SP2 (Carl Zeiss, Germany). RESULTS AND DISCUSSION Effect of protein kinase inhibitors on growth and morphology of primary roots of the A. thaliana seed lings. It was established that the growth and develop ment of primary roots were sensitive to the inhibition of phosphorylation processes in the cell by specific inhibitors of serine/threonine protein kinases. The treatment of 4daysold seedlings with all studied pro tein kinase inhibitors led to significant changes in the rate of primary root growth. As can be seen in Fig. 1, the treatment of seedlings with olomoucine or W7 for 24 h produced a decrease in the root growth rate in four time, while treatment with substance H7 or stau rosporine decreased the growth rate in 2 and 1.5, respectively, as compared to the control. It should be noticed that 24–48 h after the beginning of olomou cine action on seedlings, the roots resumed their growth. This can indicate that this type of inhibitor of cyclindependent protein kinases induced a tempo rary inhibitory effect on root growth. The effects of olomoucine that we observed on the A. thaliana seed CELL AND TISSUE BIOLOGY

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Fig. 2. Morphology of root apices of A. thaliana seedlings after treatment with inhibitors of protein kinases. (a) Control, (b) 100 µM olomoucine, (c) 10 µM W7, (d) 50 µM H7, (e) 50 nM staurosporine. Zones of root swelling and abnormal formation of root hairs are indicated with arrows. Scale: 200 µm.

lings correspond to the results of a previous study that showed that other inhibitors of cyclindependent pro tein kinases (bohemin and roskovitin) also cause the shortterm inhibition of dividing cells of the cell cycle phases G2/M of meristematic cells of the Vicia faba root apices (Binarova et al., 1998). It was also found that treatment of the A. thaliana roots with all tested protein kinase inhibitors led to sig nificant disturbances of the primary root morphology, such as the appearance of swelling in the elongation zone (Fig. 2c) and in the meristematic zone of the root (Fig. 2e) as well as changed growth and formation of the root hairs (Fig. 2b–2e). Treatment of seedlings with inhibitor W7 at a concentration of 10 μM for 24 h has been shown to lead to the abnormal formation of new root hairs in the close vicinity to the meristematic root zone (Fig. 2c). The treatment of seedlings with olomoucine at a concentration of 100 μM for 24 h (Fig. 2b), with H7 at a concentration of 50 μM (Fig. 2d), or with staurosporine at a concentration of 50 nM (Fig. 2e) induced the formation of a high num

ber of new root hairs compared to untreated seedlings (Fig. 2a). However, it should be noted that the elonga tion of newly formed root hairs in seedlings was inhib ited (as compared with control) as a result of the effect of the inhibitors mentioned above. The summarized results on the effects of different types of protein kinase inhibitors on the growth and morphology of the primary roots are presented in Table 1. Our results are in accordance with the data of another work that demonstrated that the growth of Arabidopsis roots is significantly decreased in the pres ence of inhibitors of cAMPdependent protein kinase, protein kinase C, and an inhibitor of myosin light chains (Baskin and Wilson, 1997). In particular, it was found that six different types of protein kinase inhibi tors (4dimethylaminopurine, cheleritrin, ML9, K252a, H89, and staurosporine) decelerated the growth of the primary Arabidopsis roots after the 48h treatment on a solid medium, while three of them (K252a, H89, and staurosporine) stimulated root swelling. The authors found that all of the mentioned

Table 1. Effects of different types of protein kinase inhibitors on growth and morphology of the primary roots of A. thaliana Inhibition of primary root growth Inhibitor

Olomoucine W7 H7 Staurosporine

24 h

48 h

in 4 times in 4 times in 2 times in 1.5 times

in 2.5 times in 4 times in 2 times in 1.7 times

Swelling mer elonga istematic tion zone zone – – – +

– + – –

Effect on root hairs induc growth tion of forma stimula inhibi tion tion tion + + + +

– – – –

+ + + +

Note: “+” indicates changes compared to the control; “–” indicates no changes compared to the control. CELL AND TISSUE BIOLOGY

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morphology swelling – – – –

branch bending ing – – – –

– – – –

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Effect of protein kinase inhibitors on microtubule organization in A. thaliana root cells. Since it is known that disturbances of the general morphology of root apices can result from changes in the structuralfunc tional properties of microtubules, at the next stage of the work we studied the changes in the organization of microtubules in root cells under the effect of inhibitors of various protein kinases. The detailed characteristics of the initial orientation of microtubules in cells of all growth zones of the A. thaliana root (Verbelen et al., 2006) was described in detail in our previous paper (Yemets et al., 2009). The microtubule organization in cells of the Arabidopsis control roots is presented in Fig. 3a–3d). (a)

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Fig. 3. Organization of microtubules in cells of A. thaliana roots. (a, b) Meristematic zone; (c) elongation zone; (d) differentiation zone. Arrows indicate cortical (a, c, d) and endoplasmic (b) microtubules. Scale: 20 µm.

above of inhibitors affect the process of cell stretching by causing its deceleration (Baskin and Wilson, 1997). In the present work, we demonstrated that inhibitors of Ca2+calmodulindependent and cyclindependent protein kinases reduced the primary root growth and induced swelling in various root growth zones, as well as disturbed the processes of the formation and growth of root hairs. Recently, we showed that the inhibitor of protein phosphatase 1 and protein phosphatase 2A (okadaic acid) also produces changes in the total mor phology of primary roots of A. thaliana seedlings, which specifically leads to the disturbance of root hair growth and morphology (Sheremet et al., 2009). Hence, by summarizing our obtained data, we can suggest that the inhibition of the Ca2+calmodulin dependent and cyclindependent protein kinases, as well as protein kinase C, affects the primary root growth and causes changes in the total root morphol ogy, particularly by inducing the formation of new root hairs.

As a result of all performed experiments, it was established that the treatment of seedlings with olo moucine at a concentration of 50 μM did not change the orientation of either mitotic or cortical microtu bules. An increase in this inhibitor concentration to 100 μM was accompanied after as little as 6 h by the insignificant disorientation of microtubules in epider mal cells of the transitional zone and the rootelonga tion zone. An increase in the time of olomoucine treatment to 24 or 48 h led to a change in the initial orientation of cortical microtubules from the trans verse or oblique to main root axis to the chaotic or lon gitudinal orientation in both the transitional zone and zones of elongation and differentiation (Fig. 4b, 4c, 4e). At the same time, in the meristematic root zone, the cortical and endoplasmic microtubules preserved their native organization and orientation (Fig. 4a, 4d) compared to the control (Fig. 3a, 4b). Olomoucine, an analog of purine, is known to spe cifically inhibit cyclindependent protein kinases (Vesely et al., 1994). In plant cells, several types of cyclindependent protein kinases are identified depending on structural similarity, including CDKA (classic), CDKB, and CDKD (Joubes et al., 2000). It was shown that cyclindependent protein kinases, specifically CDKA, are important for the progression of the cell cycle and entrance of plant cells into mitosis (Hemerly et al., 1995). Previously, it was found that in both animal and plant cells olomoucine inhibited the S and G2 progression of cellcycle phases G1 M (Glab et al., 1994; Meijer, 1996; Binarova et al., 1998). Several authors have established that one of the classicaltype cyclindependent protein kinases, cdc 2, is localized not only in the nucleus and on mitotic chromosomes (Bogre et al., 1997), but also with mitotic (Colasanti et al., 1993, Mineyuki, 1999; Mészáros et al., 2000) and cortical arrangements of microtubules in the plant cells (Hemsley et al., 2001). On animal cells it was revealed that the cyclindepen dent protein kinase Cdk1 catalyses phosphorylation of not only several proteins associated with microtu bules, but also of 172 serine residue of βsubunit of tubulin (FourestLeievin et al., 2006). CELL AND TISSUE BIOLOGY

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Fig. 5. Organization of microtubules in cells of A. thaliana roots after treatment with inhibitor W7 (10 µM) for 24 h. (a) Meristematic zone; (b) differentiation zone, 24 h. Arrows indicate cortical microtubules. Scale: 20 µm.

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Fig. 4. Organization of microtubules in cells of A. thaliana roots after treatment with olomoucine (100 µM). (a) Mer istematic zone, 24 h; (b) elongation zone, 24 h; (c) differ entiation zone, 24 h; (d) meristematic zone, 48 h; (e) dif ferentiation zone, 48 h. Arrows indicate cortical microtu bules. Scale: 20 µm.

In the present work we showed for the first time that under in vivo conditions, olomoucine, an inhibitor of cyclindependent protein kinases, affected the organi zation of cortical microtubules in various root cells by causing a change in their initial transverse orientation to longitudinal. Since it is known that cyclindepen dent protein kinases in the plant cell are physically associated with mitotic and cortical microtubules and the microtubule organization is sensitive to the action of olomoucine, it can be concluded that the cyclin dependent protein kinases participate in the regula tion of the organization of plant microtubules. The treatment of A. thaliana seedlings with another inhibitor, W7, also led to changes in orientation of cor tical microtubules. At a concentration of 1 μM W7 did not cause disturbances in the organization of cortical microtubules of root cells. After an increase in the W7 concentration up to 10 μM we found that, after a 6 h CELL AND TISSUE BIOLOGY

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treatment, the cortical microtubules in some epider mal cells and in cortex cells of the root elongation zone changed the initial orientation from transverse to cha otic. The longterm root treatment (24 h) with this inhibitor at the indicated concentration led to a change in the transverse (chaotic) orientation of corti cal microtubules to the longitudinal orientation in cells of both the transition zone and zones of elonga tion and differentiation (Fig. 5b). It should be noted that along with the observed disorientation of micro tubules in cells of elongation and differentiation zones, in cells of the root meristematic zone the corti cal microtubules preserved their initial orientation (Fig. 5a). An increase in the W7 concentration to 50 μM led to the complete depolymerization of microtubules in cells of all root growth zones after as little as 1 h of treatment. It is known that W7 is a specific inhibitor of Ca2+ calmodulindependent protein kinases; it is bound to two domains of the calmodulin and blocks its binding to other molecules (Osawa et al., 1998). Calmodulin is a universal Ca2+binding protein and primary intracel lular Ca2+signal receptor; it transmits signals due to the modulation of the activity of calmodulinbound proteins and generates the cell’s physiological responses to various stimuli (Trewavs and Malho, 1998; Chin and Means, 2000; Park et al., 2004). The phosphorylation of Ca2+calmodulindependent pro tein is a key mechanism for enhancing and spreading signals mediated by Ca2+calmodulin and participates in the regulation of a large spectrum of processes of plant development (Sathyanarayanan and Poovaiah, 2004; Tirichine et al., 2006). At present in Arabidopsis, several Ca2+dependent protein kinase families have been identified (Wang et al., 2007).

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In plant cells calmodulin is known to be of help in stabilizing microtubules by protecting them from the depolymerizing action of the Ca2+calmodulin com plex (Fisher et al., 1996). Due to interaction with calmodulin molecules, W7 is known to produce a destabilizing action on microtubules of protoplasts of the carrot suspensional culture (Fisher et al., 1996). On the system of N. plumbaginifolia permeabilized protoplasts we demonstrated that both subunits of plant tubulin are intensively phosphorylated following the activation of Ca2+calmodulindependent protein kinases (Blume et al., 2008a). It is also revealed that the treatment of root apices of 3daysold Allium cepa seedlings with a calmodulin antagonist, trifluoropip erazine, induces the reorganization of microtubules by changing their orientation from transverse to longitu dinal (Blume et al., 2008a). In the present work for the first time, under in vivo conditions, we showed that the W7 inhibitor changed the organization of microtubules in plant cells by action similar to that of trifluoropiperazine by produc ing a change in the initial microtubule orientation (transverse or oblique) in cells of the elongation and differentiation zones to the longitudinal orientation. The obtained data confirm the participation of Ca2+ calmodulindependent protein kinases in the regula tion of the structure of plantcell microtubules. In the course of subsequent experiments it was found that the inhibition of protein kinase C also led to changes in the organization of plant cell microtubules. The treatment of roots with the H7 inhibitor revealed the concentration–time dependence of its effect on microtubule orientation in the A. thaliana root cells. The treatment of seedlings with this inhibitor at 10 μM concentration for 3–12 h did not cause disturbances in the microtubule orientation in all zones of root growth. An increase in the treatment time to 24 h led to insignificant microtubule disorientation in epider mal cells of the root transitional zones, as well as the elongation zone of the root compared to the control. An increase in the H7 concentration to 50 μM pro duced the cortical microtubule disorientation in cells of transitional zones and the root elongation zone after 6 h of treatment of seedlings. After prolonged (24 h) treatment with H7 (50 μM) partially destroyed micro tubules were detected in cells of the root cap (Fig. 6a); in most epidermal cells of the elongation zone micro tubules were disoriented (Fig. 6b). However, it should be noted that microtubules in the meristematic root cells, as well as in trichoblasts and atrichoblasts of the root differentiation zone, were characterized by lower sensitivity to this inhibitor action (Fig. 6c). However, in the case of treating seedlings with 100 μM H7 the disorientation of cortical microtubules in epidermal cells of the root elongation zone could be observed only after 3 h of exposure. After 24 h treat ment with the inhibitor at this concentration, micro tubules were stabilized (Fig. 6d), cortical and endo

plasmic microtubules in the root meristematic zone were almost depolymerized (Fig. 6e), and cortical microtubules on cells of the elongation and differenti ation zones were thickened. In some epidermal cells cortical microtubules were disoriented and, as a result, acquired a swollen shape (Fig. 6f). Staurosporine at a concentration of 10 nM caused disturbances in the organization of the root cell micro tubules, but only after 12 h of treatment. Microtubules in cells of the transitional zone and the root elongation zone, where microtubules changed their initial trans verse to the chaotic orientation, were the most sensi tive to this inhibitor. It was revealed that after pro longed (24 h) treatment with inhibitor at this concen tration microtubules in some cells of the root transitional zone were depolymerized (Fig. 7a), as well as microtubules in both the elongation (Fig. 7b) and differentiation zones (Fig. 7c) were disoriented. The treatment of seedlings with staurosporine at a higher concentration (50 nM) for 3 h led to changes in the initial orientation of cortical microtubules in epider mal cells of the root elongation zone from transverse to chaotic (Fig. 7d). An increase in the time of treatment of seedlings to 12–24 h resulted in a disturbance of the native microtubule organization in cells of all root zones. Microtubules in the rootcap cells were partly depolymerized and, in meristematic cells and cortex cells of transition zone (Fig. 7e), as well as the elonga tion zone, disoriented and thickened microtubules were preserved. In cells of both elongation and differ entiation zones, only some thickened microtubules were visualized (Fig. 7f), since most of the microtu bules in cells of this root growth zones were depoly merized. In eukaryotic cells, protein kinase C is known to play a key role in the work of the signal system through the participation in protein phosphorylation (Stone, Walker 1995). Protein kinase C is expressed in almost all mammalian cells and its substrates in different types of cells are nuclear proteins, cytoskeleton pro teins, and enzymes (Nishizuka, 2003; Battaini and MochlyRosen, 2007). At present, analogs of protein kinase C have been found and characterized in differ ent types of plant cells, such as Brassica campestris (Nanmori et al., 1994), Oryza sativa (Karibe et al., 1995), and Solanum tuberosum (Subramaniam et al., 1997). Previously, we showed that polymyxin B, a specific inhibitor of protein kinase C, causes a change in the initial transverse microtubule orientation to longitudi nal in apex cells in 3daysold seedlings of A. cepa (Blume et al., 2008a). In the present work, for the first time, we studied the effects of other types of protein kinase C inhibitors (H7 and staurosporine) on the organization of microtubules in plant cells in vivo. Comparing the microtubule orientation in roots of untreated seedlings and of those exposed in the pres ence of protein kinase C inhibitors, a particular sensi CELL AND TISSUE BIOLOGY

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Fig. 6. Organization of microtubules in cells of A. thaliana roots after treatment with inhibitor H 7. (a) Meristematic zone, 50 µM, 24 h; (b) elongation zone, 50 µM, 24 h; (c) differentiation zone, 50 µM, 24 h; (d, e) meristematic zone, 100 µM, 24 h; (f) differ entiation zone, 100 µM, 24 h. Arrows indicate cortical microtubules. Scale: 20 µm.

tivity to the action of these chemicals at lower concen trations was found for microtubules in cells of transi tion and elongation root zones, where microtubules change their initial orientation from transverse to cha otic. An increase in concentration and time of treat ment with inhibitors leads to the stabilization of microtubules in cells of the meristematic root zone, as well as causes the disorientation and partial depoly merization of microtubules in cells of transition and elongation zones. Our results on the effects of stauro sporin on microtubule organizatione in Arabidopsis cells are in accordance with the data of an earlier study (Baskin and Wilson, 1997) that shows that, at a higher concentration (1 μM), staurosporine causes microtu bule disorganization and randomization in the A. thaliana root cells. The cells of the elongation and CELL AND TISSUE BIOLOGY

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differentiation zones had microtubules with higher sensitivity to the effect of the inhibitor at 1 μM con centration, whereas microtubules in the cells of the meristematic zone of the root were less sensitive to staurosporine (Baskin and Wilson, 1997). Thus, in the present work, we studied the effects of different specific inhibitors of serine/threonine pro tein kinases on the organization of microtubules in various types of cells in the main zones of A. thaliana root. The data are summarized in Table 2. Microtu bules in epidermal cells and cells of the cortex of tran sitional and elongation zones of the root, as well as microtubules in trichoblasts and atrichoblasts of the differentiation zone, were the most sensitive to the action of all tested types of protein kinases inhibitors. It should be noted that this differential sensitivity of

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Fig. 7. Organization of microtubules in cells of A. thaliana roots after treatment with staurosporine. (a) Meristematic zone, 10 nM, 24 h; (b) elongation zone, 10 nM, 24 h; (c) differentiation zone, 10 nM, 24 h; (d) elongation zone, 50 nM, 3 h; (e) mer istematic and transitional zones, 50 nM, 24 h; (f) differentiation zone, 50 nM, 24 h. Arrows indicate cortical microtubules. Scale: 20 µm.

microtubules in cells of different root zones to the inhibitor action is not associated with differences in their degree of accessibility to root cells, since experi ments were performed with different time intervals, i.e., both short (1 h) and longer (48 h) intervals, which is sufficient for the complete penetration of substances into all studied tissue and cells. The observed changes in microtubule orientation in root cells agree with changes in the total morphol ogy of primary roots. It has been established that the induction of the formation of a greater amount of new

root hairs after treatment with protein kinase inhibi tors could also be a consequence of the disturbance of the native microtubule organization in the cells of root growth zones. The randomization of microtubules in the cells of the differentiation zone is known to be nec essary for the formation of new root hairs (Takahashi et al., 2003). It has also been shown that microtubules participate in the regulation of the direction and sta bility of their apical growth (Sieberer et al., 2002). It should be noted that treatment with inhibitors of serine/threonine protein kinases did not cause CELL AND TISSUE BIOLOGY

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Table 2. Scheme of changes in orientation and organization of cortical microtubules in cells of the A. thaliana primary roots relative to main root axis after action of protein kinase inhibitors Meristematic zone Inhibitor rootcap

epider mis

mer istema

Transitional zone epider mis

cortex

Elongation zone epider mis

cortex

Differentiation zone atricho blasts

tricho root hairs blasts

– (Control)

Olomoucine

W7

H7

Staurospo rine

changes in microtubule organization in root hairs as compared to the control. Therefore, analyzing the obtained data, it can be suggested that the phosphory lation of microtubule proteins, including tubulin, might take a mediated part in the process of the induc tion and formation of new root hairs and produce no effect on their further growth. In turn, according to the data obtained by us previ ously about the effect of okadaic acid on microtubule organization in the A. thaliana root cells, it was estab lished that the induction of protein hyperphosphory lation as a result of the inhibition of protein phos phatase 1 and protein phosphatase 2A leads to differ ent effects on the microtubule organization depending on the type of the cells, which cause either the depoly merization of cortical microtubules in the cells of the elongation zone or microtubule stabilization in the cells of the differentiation zone (Sheremet et al., 2009). Microtubules in root hairs were particularly sensitive to the action of okadaic acid at all studied concentrations compared to the control; as a result, the root hairs changed their general morphology, i.e., they were bent, branched, swelled, or stopped their growth completely. By summarizing the obtained data, it can be sug gested that the change in the initial microtubule orien tation in cells of elongation and differentiation zones of the root due to the inhibition of various types of serine/threonine protein kinases may be the cause of change in their growth rate, as well as the disturbance of general morphology of the primary root and root hair formation. The inhibition of serine/threonine protein phosphatases produces the opposite effect on CELL AND TISSUE BIOLOGY

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both the growth and general morphology of the pri mary root and the organization of microtubules in var ious types of cells of all root growth zones (Sheremet et al., 2009). Hence, it can be concluded that the phosphorylation of microtubules proteins, primarily tubulin, on serine and threonine residues could be involved in regulation of processes of root elongation as well as participates in processes of the induction, formation, and growth of root hairs. This in turn indi cates that this type of posttranslational modification participates in the regulation of higher plant develop ment. REFERENCES Baskin, T.I. and Wilson, J.E., Inhibitors of Protein Kinases and Phosphatases Alter Root Morphology and Disorganize Cortical Microtubules, Plant Physiol., 1997, vol. 113, pp. 493–502. Battaini, F. and Mochly–Rosen, D., Happy Birthday Pro tein Kinase C: Past, Present and Future of a Superfamily, Pharmacol. Res., 2007, vol. 55, pp. 461–466. BenNissan, G., Cui, W., Kim, D.J., Yang, Y., Yoo, B.C., and Lee, J.Y., Arabidopsis Casein Kinase 1Like 6 Contains a MicrotubuleBinding Domain and Affects the Organiza tion of Cortical Microtubules, Plant Physiol., 2008, vol. 148, pp. 1897–1907. Binarova, P., Dolezel, J., Draber, P., HerberleBors, E., Stnad, M., Bogre, L., Treatment of Vicia faba Root Tip Cells with Specific Inhibitors to CyclinDependent Kinases Leads to Abnormal Spindle Formation, Plant J., 1998, vol. 16, pp. 697–707. Blume, Ya., Yemets, A., Sulimenko, V., Sulimenko, T., Chan, J., Lloyd, C., and Draber, P., Evidence of Tyrosine

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