effects of inhibitors of serine/threonine protein kinases on arabidopsis thaliana root morphology...

ISSN 1990�519X, 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.

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Abbreviations used: cAMP, cyclic adenosine mono�phosphate; DMSO, dimethylsulfoxide; CDK, cyclin�dependent kinase; GFP, green fluorescent protein;MAP, microtubule�associated 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 tyrosinekinases and tyrosine phosphatases on the model plantArabidopsis thaliana expressing the gfp�map4 gene, wehave found that phosphorylation on tyrosine residuesinvolved in the organization of microtubules in differ�ent types of cells in all growth zones of the primaryroot (Yemets et al., 2008). Recently, in plant cells, thecasein�like kinase, CKL6, was revealed, which cata�lyzes the phosphorylation of serine residues of thetubulin β�subunit at positions 413 and 420 (Ben–Nis�san et al., 2008). In another work, it was demonstratedthat α�tubulin from the tea Camellia sinensis had foursites of phosphorylation by protein kinase C (Fanget al., 2006). Recently, 27 phosphorylated proteinswere isolated from the suspension culture of Nicotiana

tabacum; tubulin phosphorylated by the Ca2+�calm�odulin�dependent process was also identified amongthese proteins (Gerber et al., 2006). However, thefunctional role of the posttranslational phosphoryla�tion of microtubules proteins on serine and threonineresidues has hardly been investigated.

At present, the main types of serine and threonineprotein kinases associated with plant microtubules (byanalogy with animal cell) have been characterized(Karpov et al., 2009). Hence, it can be suggested thatthis posttranslational modification plays an importantrole in the organization of the cortical microtubulenetwork and the regulation of their dynamic propertiesin plant cells.

On the other hand, we have established that bothtubulin subunits in plant cells are intensively phospho�rylated on serine and threonine residues as a result ofthe activation of the corresponding protein kinases(cAMP�dependent, Ca2+, Ca2+�calmodulin), as wellas protein kinase C (Blume et al., 2008a). It was foundthat the use of specific inhibitors of Ca2+�calmodulin�dependent protein kinases and protein kinase C led tochanges in the microtubule organization in root apexcells of 3�day�old seedlings of Allium cepa (Blumeet al., 2008a). In another work, we showed that theinduction of protein hyperphosphorylation by the

Effects of Inhibitors of Serine/Threonine Protein Kinases on Arabidopsis thaliana Root Morphology and Microtubule

Organization in Its CellsYa. 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, Ukraineb University of Antwerp, Antwerp, Belgium

*e�mail: yarasheremet@gmail.ñomReceived June 9, 2009

Abstract—The effect of different types of serine/threonine protein kinase inhibitors (cyclin�dependent,Ca2+�calmodulin dependent and protein kinase C) on the microtubule organization in cells of Arabidopsisthaliana main primary root zones were investigated in vivo. It was found that the microtubules in epidermaland cortex cells of transition and elongation zones, as well as microtubules in trichoblasts and atrichoblastsof 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 ofprimary root zones under the effect of serine/threonine protein kinase inhibitors led to further disturbancesin 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

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inhibition of serine/threonine protein phosphatasesalso leads to the disturbance of the microtubule orga�nization in plant cells (Sheremet et al., 2009).

Thus, to elucidate the role of different types ofserine/threonine protein kinases in the phosphoryla�tion of proteins of plant cell microtubules and theirfunctional significance, we have attempted to studythe effects of inhibitors of several serine/threonineprotein kinases on the rearrangement of microtubulesin different types of cells. In the present work, wedetermined the interconnection between inhibition ofactivities of serine/threonine protein kinases (such ascyclin�dependent, Ca2+�calmodulin�dependent pro�tein kinase and protein kinase C) and the change in thegrowth parameters and morphology of the primaryroots, 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 gfp�map4 (Mathur and Chua, 2000) wereused in current study. In experiments with inhibitors ofserine/threonine protein kinases, we used 4�days�oldArabidopsis 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. Aninhibitor of Ca2+�calmodulin�dependent proteinkinases W7 (Sigma, US) was dissolved in distilledwater at +70°C and the stock solution (100 mM) wasstored 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 workingsolutions of inhibitors at various concentrations wereprepared in distilled water. The final DMSO concen�tration in these solutions did not exceed 0.5% and, aswe established previously, is not toxic for plant cellsand microtubules (Mitrofanova et al., 2003; Yemets etal., 2005; Ozheredov et al., 2009), which is in agree�ment with the results of previous works.

In the first series of experiments, we studied theeffect of protein kinase inhibitors at various concen�trations for 6, 24, and 48 h on the growth and mor�phology of roots of 4�days�old A. thaliana seedlings.The obtained data were documented using a CanonPower Shot G6 digital camera in the regime ofmicroshooting. The length of the A. thaliana roots wasmeasured by the previously described procedure(Yemets et al., 2008). All experiments were performedin three repeats and the results were processed statisti�cally by the commonly accepted methods (Lakin,1990).

To study the short�term and prolonged effects ofspecific inhibitors on the organization and orientationof microtubules in cells of various growth zones of theprimary root, 4�days�old seedlings were treated withthe corresponding substances at different concentra�tions for 1–48 h. The GFP�labeled microtubules inA. thaliana cells were visualized in vivo by using con�focal laser scanning microscope LSM 510 META(Carl Zeiss, Germany). To obtain the three�dimen�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× (Plan�Apochromat). Based on serial opticalsections (with the 0.3–0.5 μM interval), 3D models ofmicrotubules were reconstructed using LSM 510 soft�ware, version 4.0 SP2 (Carl Zeiss, Germany).

RESULTS AND DISCUSSION

Effect of protein kinase inhibitors on growth andmorphology 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 inhibitionof phosphorylation processes in the cell by specificinhibitors of serine/threonine protein kinases. Thetreatment of 4�days�old seedlings with all studied pro�tein kinase inhibitors led to significant changes in therate of primary root growth. As can be seen in Fig. 1,the treatment of seedlings with olomoucine or W7 for24 h produced a decrease in the root growth rate infour 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 benoticed that 24–48 h after the beginning of olomou�cine action on seedlings, the roots resumed theirgrowth. This can indicate that this type of inhibitor ofcyclin�dependent protein kinases induced a tempo�rary inhibitory effect on root growth. The effects ofolomoucine that we observed on the A. thaliana seed�

4842363024181260Time, h

30

25

20

15

10

5

Accretion of length, %

ContrololomoucineW7H7staurosporine

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


EFFECTS OF INHIBITORS OF SERINE/THREONINE PROTEIN KINASES 401

lings correspond to the results of a previous study thatshowed that other inhibitors of cyclin�dependent pro�tein kinases (bohemin and roskovitin) also cause theshort�term inhibition of dividing cells of the cell cyclephases G2/M of meristematic cells of the Vicia fabaroot apices (Binarova et al., 1998).

It was also found that treatment of the A. thalianaroots with all tested protein kinase inhibitors led to sig�nificant disturbances of the primary root morphology,such as the appearance of swelling in the elongationzone (Fig. 2c) and in the meristematic zone of the root(Fig. 2e) as well as changed growth and formation ofthe root hairs (Fig. 2b–2e). Treatment of seedlingswith inhibitor W7 at a concentration of 10 μM for 24 hhas been shown to lead to the abnormal formation ofnew root hairs in the close vicinity to the meristematicroot zone (Fig. 2c). The treatment of seedlings witholomoucine 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 of50 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 effectof the inhibitors mentioned above. The summarizedresults on the effects of different types of proteinkinase inhibitors on the growth and morphology of theprimary roots are presented in Table 1.

Our results are in accordance with the data ofanother work that demonstrated that the growth ofArabidopsis roots is significantly decreased in the pres�ence of inhibitors of cAMP�dependent protein kinase,protein kinase C, and an inhibitor of myosin lightchains (Baskin and Wilson, 1997). In particular, it wasfound that six different types of protein kinase inhibi�tors (4�dimethylaminopurine, cheleritrin, ML�9,K252a, H�89, and staurosporine) decelerated thegrowth of the primary Arabidopsis roots after the 48�htreatment on a solid medium, while three of them(K252a, H�89, and staurosporine) stimulated rootswelling. The authors found that all of the mentioned

(а) (b) (c) (d) (e)

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 formationof root hairs are indicated with arrows. Scale: 200 µm.

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

Inhibitor

Inhibition of primary root growth

Swelling Effect on root hairs

mer�istematic

zone

elonga�tion zone

induc�tion of forma�

tion

growth morphology

24 h 48 h stimula�tion

inhibi�tion swelling branch�

ing bending

Olomoucine in 4 times in 2.5 times – – + – + – – –

W7 in 4 times in 4 times – + + – + – – –

H7 in 2 times in 2 times – – + – + – – –

Staurosporine in 1.5 times in 1.7 times + – + – + – – –

Note: “+” indicates changes compared to the control; “–” indicates no changes compared to the control.

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above of inhibitors affect the process of cell stretchingby causing its deceleration (Baskin and Wilson, 1997).In the present work, we demonstrated that inhibitorsof Ca2+�calmodulin�dependent and cyclin�dependentprotein kinases reduced the primary root growth andinduced swelling in various root growth zones, as wellas disturbed the processes of the formation and growthof root hairs. Recently, we showed that the inhibitor ofprotein 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 hairgrowth and morphology (Sheremet et al., 2009).Hence, by summarizing our obtained data, we cansuggest that the inhibition of the Ca2+�calmodulin�dependent and cyclin�dependent protein kinases, aswell as protein kinase C, affects the primary rootgrowth and causes changes in the total root morphol�ogy, particularly by inducing the formation of new roothairs.

Effect of protein kinase inhibitors on microtubuleorganization in A. thaliana root cells. Since it is knownthat disturbances of the general morphology of rootapices can result from changes in the structural�func�tional properties of microtubules, at the next stage ofthe work we studied the changes in the organization ofmicrotubules in root cells under the effect of inhibitorsof various protein kinases. The detailed characteristicsof the initial orientation of microtubules in cells of allgrowth 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 incells of the Arabidopsis control roots is presented inFig. 3a–3d).

As a result of all performed experiments, it wasestablished that the treatment of seedlings with olo�moucine at a concentration of 50 μM did not changethe orientation of either mitotic or cortical microtu�bules. An increase in this inhibitor concentration to100 μM was accompanied after as little as 6 h by theinsignificant disorientation of microtubules in epider�mal cells of the transitional zone and the root�elonga�tion zone. An increase in the time of olomoucinetreatment to 24 or 48 h led to a change in the initialorientation 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 andzones of elongation and differentiation (Fig. 4b, 4c,4e). At the same time, in the meristematic root zone,the cortical and endoplasmic microtubules preservedtheir 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 cyclin�dependent protein kinases(Vesely et al., 1994). In plant cells, several types ofcyclin�dependent protein kinases are identifieddepending on structural similarity, including CDK�A(classic), CDK�B, and CDK�D (Joubes et al., 2000).It was shown that cyclin�dependent protein kinases,specifically CDK�A, are important for the progressionof the cell cycle and entrance of plant cells into mitosis(Hemerly et al., 1995). Previously, it was found that inboth animal and plant cells olomoucine inhibited theprogression of cell�cycle phases G1 S and G2 M (Glab et al., 1994; Meijer, 1996; Binarova et al.,1998). Several authors have established that one of theclassical�type cyclin�dependent protein kinases,cdc 2, is localized not only in the nucleus and onmitotic chromosomes (Bogre et al., 1997), but alsowith mitotic (Colasanti et al., 1993, Mineyuki, 1999;Mészáros et al., 2000) and cortical arrangements ofmicrotubules in the plant cells (Hemsley et al., 2001).On animal cells it was revealed that the cyclin�depen�dent protein kinase Cdk1 catalyses phosphorylation ofnot only several proteins associated with microtu�bules, but also of 172 serine residue of β�subunit oftubulin (Fourest�Leievin et al., 2006).

(a) (b)

(c) (d)

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



In the present work we showed for the first time thatunder in vivo conditions, olomoucine, an inhibitor ofcyclin�dependent protein kinases, affected the organi�zation of cortical microtubules in various root cells bycausing a change in their initial transverse orientationto longitudinal. Since it is known that cyclin�depen�dent protein kinases in the plant cell are physicallyassociated with mitotic and cortical microtubules andthe microtubule organization is sensitive to the actionof 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 anotherinhibitor, W7, also led to changes in orientation of cor�tical microtubules. At a concentration of 1 μM W7 didnot cause disturbances in the organization of corticalmicrotubules of root cells. After an increase in the W7concentration up to 10 μM we found that, after a 6 h

treatment, the cortical microtubules in some epider�mal cells and in cortex cells of the root elongation zonechanged the initial orientation from transverse to cha�otic. The long�term root treatment (24 h) with thisinhibitor at the indicated concentration led to achange in the transverse (chaotic) orientation of corti�cal microtubules to the longitudinal orientation incells of both the transition zone and zones of elonga�tion and differentiation (Fig. 5b). It should be notedthat along with the observed disorientation of micro�tubules in cells of elongation and differentiationzones, in cells of the root meristematic zone the corti�cal microtubules preserved their initial orientation(Fig. 5a). An increase in the W7 concentration to50 μM led to the complete depolymerization ofmicrotubules in cells of all root growth zones after aslittle as 1 h of treatment.

It is known that W7 is a specific inhibitor of Ca2+�calmodulin�dependent protein kinases; it is bound totwo domains of the calmodulin and blocks its bindingto other molecules (Osawa et al., 1998). Calmodulin isa universal Ca2+�binding protein and primary intracel�lular Ca2+�signal receptor; it transmits signals due tothe modulation of the activity of calmodulin�boundproteins and generates the cell’s physiologicalresponses to various stimuli (Trewavs and Malho,1998; Chin and Means, 2000; Park et al., 2004). Thephosphorylation of Ca2+�calmodulin�dependent pro�tein is a key mechanism for enhancing and spreadingsignals mediated by Ca2+�calmodulin and participatesin the regulation of a large spectrum of processes ofplant development (Sathyanarayanan and Poovaiah,2004; Tirichine et al., 2006). At present in Arabidopsis,several Ca2+�dependent protein kinase families havebeen identified (Wang et al., 2007).

(a) (b)

(d) (e)

(c)

Fig. 4. Organization of microtubules in cells of A. thalianaroots 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.

(a) (b)

Fig. 5. Organization of microtubules in cells of A. thalianaroots 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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In plant cells calmodulin is known to be of help instabilizing microtubules by protecting them from thedepolymerizing action of the Ca2+�calmodulin com�plex (Fisher et al., 1996). Due to interaction withcalmodulin molecules, W7 is known to produce adestabilizing action on microtubules of protoplasts ofthe carrot suspensional culture (Fisher et al., 1996).On the system of N. plumbaginifolia permeabilizedprotoplasts we demonstrated that both subunits ofplant tubulin are intensively phosphorylated followingthe activation of Ca2+�calmodulin�dependent proteinkinases (Blume et al., 2008a). It is also revealed thatthe treatment of root apices of 3�days�old Allium cepaseedlings with a calmodulin antagonist, trifluoropip�erazine, induces the reorganization of microtubules bychanging their orientation from transverse to longitu�dinal (Blume et al., 2008a).

In the present work for the first time, under in vivoconditions, we showed that the W7 inhibitor changedthe organization of microtubules in plant cells byaction similar to that of trifluoropiperazine by produc�ing a change in the initial microtubule orientation(transverse or oblique) in cells of the elongation anddifferentiation zones to the longitudinal orientation.The obtained data confirm the participation of Ca2+�calmodulin�dependent protein kinases in the regula�tion of the structure of plant�cell microtubules.

In the course of subsequent experiments it wasfound that the inhibition of protein kinase C also led tochanges in the organization of plant cell microtubules.The treatment of roots with the H7 inhibitor revealedthe concentration–time dependence of its effect onmicrotubule orientation in the A. thaliana root cells.The treatment of seedlings with this inhibitor at 10 μMconcentration for 3–12 h did not cause disturbances inthe microtubule orientation in all zones of rootgrowth. An increase in the treatment time to 24 h ledto insignificant microtubule disorientation in epider�mal cells of the root transitional zones, as well as theelongation zone of the root compared to the control.An increase in the H7 concentration to 50 μM pro�duced the cortical microtubule disorientation in cellsof transitional zones and the root elongation zone after6 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 shouldbe noted that microtubules in the meristematic rootcells, as well as in trichoblasts and atrichoblasts of theroot differentiation zone, were characterized by lowersensitivity to this inhibitor action (Fig. 6c).

However, in the case of treating seedlings with100 μM H7 the disorientation of cortical microtubulesin epidermal cells of the root elongation zone could beobserved 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 zonewere almost depolymerized (Fig. 6e), and corticalmicrotubules on cells of the elongation and differenti�ation zones were thickened. In some epidermal cellscortical microtubules were disoriented and, as a result,acquired a swollen shape (Fig. 6f).

Staurosporine at a concentration of 10 nM causeddisturbances in the organization of the root cell micro�tubules, but only after 12 h of treatment. Microtubulesin cells of the transitional zone and the root elongationzone, 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 roottransitional zone were depolymerized (Fig. 7a), as wellas microtubules in both the elongation (Fig. 7b) anddifferentiation zones (Fig. 7c) were disoriented. Thetreatment of seedlings with staurosporine at a higherconcentration (50 nM) for 3 h led to changes in theinitial orientation of cortical microtubules in epider�mal cells of the root elongation zone from transverse tochaotic (Fig. 7d). An increase in the time of treatmentof seedlings to 12–24 h resulted in a disturbance of thenative microtubule organization in cells of all rootzones. Microtubules in the root�cap cells were partlydepolymerized and, in meristematic cells and cortexcells of transition zone (Fig. 7e), as well as the elonga�tion zone, disoriented and thickened microtubuleswere preserved. In cells of both elongation and differ�entiation zones, only some thickened microtubuleswere 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 toplay a key role in the work of the signal system throughthe participation in protein phosphorylation (Stone,Walker 1995). Protein kinase C is expressed in almostall mammalian cells and its substrates in differenttypes of cells are nuclear proteins, cytoskeleton pro�teins, and enzymes (Nishizuka, 2003; Battaini andMochly�Rosen, 2007). At present, analogs of proteinkinase 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 specificinhibitor of protein kinase C, causes a change in theinitial transverse microtubule orientation to longitudi�nal in apex cells in 3�days�old seedlings of A. cepa(Blume et al., 2008a). In the present work, for the firsttime, we studied the effects of other types of proteinkinase C inhibitors (H7 and staurosporine) on theorganization of microtubules in plant cells in vivo.Comparing the microtubule orientation in roots ofuntreated seedlings and of those exposed in the pres�ence of protein kinase C inhibitors, a particular sensi�



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 microtubuleschange their initial orientation from transverse to cha�otic. An increase in concentration and time of treat�ment with inhibitors leads to the stabilization ofmicrotubules in cells of the meristematic root zone, aswell as causes the disorientation and partial depoly�merization of microtubules in cells of transition andelongation zones. Our results on the effects of stauro�sporin on microtubule organizatione in Arabidopsiscells are in accordance with the data of an earlier study(Baskin and Wilson, 1997) that shows that, at a higherconcentration (1 μM), staurosporine causes microtu�bule disorganization and randomization in theA. thaliana root cells. The cells of the elongation and

differentiation zones had microtubules with highersensitivity to the effect of the inhibitor at 1 μM con�centration, whereas microtubules in the cells of themeristematic zone of the root were less sensitive tostaurosporine (Baskin and Wilson, 1997).

Thus, in the present work, we studied the effects ofdifferent specific inhibitors of serine/threonine pro�tein kinases on the organization of microtubules invarious types of cells in the main zones of A. thalianaroot. 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 asmicrotubules in trichoblasts and atrichoblasts of thedifferentiation zone, were the most sensitive to theaction of all tested types of protein kinases inhibitors.It should be noted that this differential sensitivity of

(a) (b) (c)

(d) (e) (f)

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.

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microtubules in cells of different root zones to theinhibitor action is not associated with differences intheir 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, whichis sufficient for the complete penetration of substancesinto all studied tissue and cells.

The observed changes in microtubule orientationin root cells agree with changes in the total morphol�ogy of primary roots. It has been established that theinduction 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 ofthe native microtubule organization in the cells of rootgrowth zones. The randomization of microtubules inthe cells of the differentiation zone is known to be nec�essary for the formation of new root hairs (Takahashiet al., 2003). It has also been shown that microtubulesparticipate in the regulation of the direction and sta�bility of their apical growth (Sieberer et al., 2002). Itshould be noted that treatment with inhibitors ofserine/threonine protein kinases did not cause

(a) (b) (c)

(d) (e) (f)

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.



changes in microtubule organization in root hairs ascompared to the control. Therefore, analyzing theobtained 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 noeffect on their further growth.

In turn, according to the data obtained by us previ�ously about the effect of okadaic acid on microtubuleorganization 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 dependingon the type of the cells, which cause either the depoly�merization of cortical microtubules in the cells of theelongation zone or microtubule stabilization in thecells of the differentiation zone (Sheremet et al.,2009). Microtubules in root hairs were particularlysensitive to the action of okadaic acid at all studiedconcentrations compared to the control; as a result,the root hairs changed their general morphology, i.e.,they were bent, branched, swelled, or stopped theirgrowth 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 zonesof the root due to the inhibition of various types ofserine/threonine protein kinases may be the cause ofchange in their growth rate, as well as the disturbanceof general morphology of the primary root and roothair formation. The inhibition of serine/threonineprotein phosphatases produces the opposite effect on

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 (Sheremetet al., 2009). Hence, it can be concluded that thephosphorylation of microtubules proteins, primarilytubulin, on serine and threonine residues could beinvolved in regulation of processes of root elongationas well as participates in processes of the induction,formation, and growth of root hairs. This in turn indi�cates that this type of post�translational modificationparticipates in the regulation of higher plant develop�ment.

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Table 2. Scheme of changes in orientation and organization of cortical microtubules in cells of the A. thaliana primary rootsrelative to main root axis after action of protein kinase inhibitors

Inhibitor

Meristematic zone Transitional zone Elongation zone Differentiation zone

root�cap epider�mis

mer�istema

epider�mis cortex epider�

mis cortex atricho�blasts

tricho�blasts root hairs

– (Control)

Olomoucine

W7

H7

Staurospo�rine

408


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effects of inhibitors of serine/threonine protein kinases on arabidopsis thaliana root morphology...

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