Cytokinesis in Fra2 Arabidopsis Thaliana P60-Katanin Mutant: Defects in Cell Plate/Daughter Wall Formation

International Journal of Molecular Sciences

Article Cytokinesis in fra2 Arabidopsis thaliana p60-Katanin Mutant: Defects in Cell Plate/Daughter Wall Formation

Emmanuel Panteris 1,* , Anna Kouskouveli 1, Dimitris Pappas 1 and Ioannis-Dimosthenis S. Adamakis 2,*

1 Department of Botany, School of Biology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece; [email protected] (A.K.); [email protected] (D.P.) 2 Section of Botany, Department of Biology, National and Kapodistrian University of Athens, 157 84 Athens, Greece * Correspondence: [email protected] (E.P.); [email protected] (I.-D.S.A.); Tel.: +30-2310-998908 (E.P.)

Abstract: Cytokinesis is accomplished in higher plants by the phragmoplast, creating and con- ducting the cell plate to separate daughter nuclei by a new cell wall. The microtubule-severing enzyme p60-katanin plays an important role in the centrifugal expansion and timely disappearance of phragmoplast microtubules. Consequently, aberrant structure and delayed expansion rate of the phragmoplast have been reported to occur in p60-katanin mutants. Here, the consequences of p60-katanin malfunction in cell plate/daughter wall formation were investigated by transmission electron microscopy (TEM), in root cells of the fra2 Arabidopsis thaliana loss-of-function mutant. In addition, deviations in the chemical composition of cell plate/new cell wall were identiﬁed by immunolabeling and confocal microscopy. It was found that, apart from defective phragmoplast microtubule organization, cell plates/new cell walls also appeared faulty in structure, being unevenly thick and perforated by large gaps. In addition, demethylesteriﬁed homogalacturonans were

prematurely present in fra2 cell plates, while callose content was signiﬁcantly lower than in the wild type. Furthermore, KNOLLE syntaxin disappeared from newly formed cell walls in fra2 earlier than

Citation: Panteris, E.; Kouskouveli, in the wild type. Taken together, these observations indicate that delayed cytokinesis, due to faulty A.; Pappas, D.; Adamakis, I.-D.S. phragmoplast organization and expansion, results in a loss of synchronization between cell plate Cytokinesis in fra2 Arabidopsis thaliana growth and its chemical maturation. p60-katanin Mutant: Defects in Cell Plate/Daughter Wall Formation. Int. Keywords: Arabidopsis thaliana; callose; cell plate; cross wall; cytokinesis; homogalacturonans; J. Mol. Sci. 2021, 22, 1405. https:// katanin; KNOLLE syntaxin; microtubules; phragmoplast doi.org/10.3390/ijms22031405

Academic Editor: Mamoru Sugita Received: 11 January 2021 1. Introduction Accepted: 28 January 2021 Published: 30 January 2021 Cytokinesis, the process by which parent cells divide after karyokinesis, is accomplished in higher plants by the function of the phragmoplast [1–3]. Typically, an array

Publisher’s Note: MDPI stays neutral of two anti-parallel microtubule groups, originally deriving by the central spindle, me- with regard to jurisdictional claims in diates the formation of a cell plate, consisting of fusing dictyosome vesicles [3,4]. These published maps and institutional affil- microtubules are perpendicular to the cell plate, with their plus ends pointing toward the iations. equatorial region [5]. Both the phragmoplast and cell plate initiate between the separated chromosome groups at telophase, then expand centrifugally to meet the parent cell wall at the cortical division site [3–5]. Cell plate is built by dictyosome-derived vesicles, the fusion of which into a unified cell plate is mediated by KNOLLE, a cytokinesis-specific plant syntaxin that, apart from being recruited to the cell plate, also persists for a while on Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. newly built cell walls [6,7]. Initially, cell plate consists mainly of callose, offering integrity This article is an open access article and rigidity, and highly methylesterified pectins, while a milestone of its maturation is distributed under the terms and the replacement of callose by cellulose and the de-esterification of pectins [8]. During cell conditions of the Creative Commons plate expansion, phragmoplast microtubules depolymerize in the center, where the cell Attribution (CC BY) license (https:// plate is stabilized, and new ones appear at the rim of the expanding cell plate, where new creativecommons.org/licenses/by/ vesicles are added [4]. After expansion outside the daughter nuclei zone and initial cell 4.0/). plate stabilization, phragmoplast microtubules are typically short, perpendicular to the

Int. J. Mol. Sci. 2021, 22, 1405. https://doi.org/10.3390/ijms22031405 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 1405 2 of 14

cell plate, eventually disappearing after the final fusion of the cell plate with parent cell walls [9]. Several proteins are involved in phragmoplast organization, expansion, and function, among which p60-katanin, a microtubule-severing enzyme that belongs in the family of AAA ATPases [10]. In particular, p60-katanin severs microtubules at their (-) ends, releasing them from their nucleation sites and allowing them to achieve their appropriate organization pattern [11]. Initially, failure in severing by p60-katanin was associated with loss of cell growth anisotropy, due to cortical microtubule disorientation, a defect observed in a variety of mutants [12–15]. Furthermore, microtubule severing by p60-katanin was found to be important in several developmental processes of plants [16], both vegetative and reproductive [17], affecting tissue patterning [18]. During cytokinesis, p60-katanin was found to be located at the (-) microtubule ends of the phragmoplast, distal to the cell plate [19], most probably mediating their release from the nuclear surface and “trimming” the expanding phragmoplast microtubules to the appropriate length and arrangement [19]. As already reported by confocal microscopy observations, failure in microtubule severing by p60-katanin, in the relevant Arabidopsis thaliana mutants, results in a “double-arrow” phragmoplast configuration: during expansion, microtubules appear elongated and bent, as their (-) ends may remain attached to the nuclear surface [20]. This structural aberration also affects the rate of cytokinesis, which in p60-katanin mutants is significantly slower than in the wild-type [21]. Although the above defects in phragmoplast organization and function are well- established, the fine structure of cytokinesis in p60-katanin mutants has not been studied yet. Therefore, in an effort to further elucidate the role of p60-katanin in cytokinesis, a detailed structural investigation of cell plate/daughter wall formation was performed in fra2 A. thaliana p60-katanin mutant by transmission electron microscopy (TEM). Moreover, the pectic components of the developing cell plate and cross wall were identified by a monoclonal antibody (JIM5), while detection of callose and the KNOLLE syntaxin was also performed by epi-fluorescence and/or confocal microscopy.

2. Results 2.1. Cell Plate Formation in the Wild-Type Wild-type (ecotype Columbia; Col-0) root cells at cytokinesis displayed typical cell plates at each stage of development (early, mid and late) [3], with the daughter nuclei being well-separated (Figures1 and2). In the above cells, after the formation of a cell plate assembly matrix at the center of the cell, cell plates, uniform in thickness (Figures1 and2), expanded centrifugally to the parental cell wall, where they fused, thus successfully completing cell division (Figure2). The nascent cross wall was also uniformly thick, interrupted only by primary plasmodesmata (Figure2). Simultaneously with cell plate maturation in the central part of the cell, phragmoplast microtubules disassembled in this area, expanding centrifugally and remaining restricted to the growing edge of the cell plate (Figure3a). Once the cell plate reached and fused with the parent cell wall, phragmoplast microtubules disappeared (Figure2).

2.2. Cell Plate Formation in fra2 While in cytokinetic root cells of the wild-type, with a growing cell plate, the microtubules of the expanding phragmoplast were short and restricted at the edges of the cell plate (Figure3a), in cytokinetic root cells of fra2, at a stage similar to that of the wild-type, phragmoplast microtubules were mainly restricted at the edges of the growing cell plate, but they were longer than those of the phragmoplasts of wild-type cells, bending and extending towards the daughter nuclei, having a “double arrow” conﬁguration at side view (Figure3b, Figure A1a), as already reported [19,20]. Int. J. Mol. Sci. 2021, 22, 1405 3 of 14 Int. J. Mol. Sci. 2021, 22, 1405 3 of 15

Figure 1. TEM micrographs of a cytokinetic wild-type root cell at central longitudinal section. (a) Lower magnification Figure 1. TEM micrographs of a cytokinetic wild-type root cell at central longitudinal section. (a) Lower magnification view of the whole cell. The daughter nuclei (N) exhibit telophase morphology, baring condensed chromatin. (b–d) Higher view of the whole cell. The daughter nuclei (N) exhibit telophase morphology, baring condensed chromatin. (b–d) Higher magnification images of the corresponding areas, defined by rectangles in (a). The cell plate (defined by arrows in a) is builtmagnification up by vesicles, images not of the yet corresponding connected with areas, the definedparent wall by rectangles at its edges, in (a where). The cellphragmoplast plate (defined microtubules by arrows incan a) isbe built ob- servedup by vesicles, (b, d, arrowheads). not yet connected Microtubules with the are parent absent wall from at its the edges, central where cell phragmoplast plate part (c). microtubules CW, cell wall. can Bars: be observed a, 5 μm; (( b–d,b,d), 200arrowheads). nm. Microtubules are absent from the central cell plate part (c). CW, cell wall. Bars: (a), 5 µm; (b–d), 200 nm.

Int. J. Mol. Sci. 2021, 22, 1405 4 of 14 Int. J. Mol. Sci. 2021, 22, 1405 4 of 15

Figure 2. TEM micrographsmicrographs ofof aa post-cytokineticpost-cytokinetic wild-type wild-type root root cell cell at at central central longitudinal longitudinal section. section. (a) ( Lowera) Lower magnification magnification view of the whole cell. The daughter nuclei (N) exhibit interphase morphology. (b–d) Higher magnification images view of the whole cell. The daughter nuclei (N) exhibit interphase morphology. (b–d) Higher magnification images of the of the corresponding areas, defined by rectangles in (a). The mature cell plate/nascent cross wall (defined by arrows in a) corresponding areas, defined by rectangles in (a). The mature cell plate/nascent cross wall (defined by arrows in (a)) is is consolidated, connected with the parent wall, exhibiting uniform width, with some plasmodesmata (arrowheads in c, consolidated,d) interrupting connected its continuity. with theSome parent remnants wall, exhibitingof phragmoplast uniform microtubules width, with somecan be plasmodesmata still observed (b, (arrowheads arrowheads). in (CW,c,d)) interruptingcell wall. Bars: its a, continuity. 5 μm; b–d, Some 200 nm. remnants of phragmoplast microtubules can be still observed ((b), arrowheads). CW, cell wall. Bars: (a), 5 µm; (b–d), 200 nm. 2.2. Cell Plate Formation in fra2 In fra2 roots, cell plates of cytokinetic cells exhibited several structural defects. Early cell platesWhile ofin mid-cytokineticcytokinetic root cellscells exhibited of the wild-type, asymmetrical, with a unilateral growing growthcell plate, (Figure the mi-4), crotubuleswhile in many of the cases expanding the phragmoplast phragmoplast microtubules were short extended and restricted towards theat the daughter edges nucleiof the cell(Figure plate4b–d, (Figure Figure 3a), A1 inc,d), cytokinetic an observation root cells in accordance of fra2, at with a stage observations similar to with that tubulin of the wild-type,immunostaining phragmoplast (see Figure microtubules3b). In addition, were inma severalinly restricted cases, phragmoplast at the edges microtubules of the grow- ingwere cell observed plate, but to penetrate they were through longer the than whole those cell of plate the width,phragmopla “protruding”sts of wild-type into theneigh- cells, bendingboring cytoplasm and extending (Figure towards A1b). Inthe post-cytokinetic daughter nuclei, cells, having cross a walls “double were arrow” discontinuous, configuration at side view (Figure 3b, Figure A(1a)), as already reported [19,20]. consisting of still growing cell plate parts (Figure5a–b 3) and unilaterally matured cross wall fragments (Figure5a 2). In some cases, the cross walls were abnormally developed, lined by aggregations of multilamellar bodies (Figure5c–c 3), most probably representing remnants of vesicle membranes. Phragmoplasts persisted in post-cytokinetic cells, exhibiting numerous microtubules (Figure5b–b 3). Moreover, post-cytokinetic cell walls exhibited extensive gaps (Figure6) or persisting phragmoplast microtubules penetrating through the newly-formed cell wall (Figure6).

Int. J. Mol. Sci. 2021, 22, 1405 5 of 14

2.3. Callose, KNOLLE, and Demethylesterified Homogalacturonan (DeSPHG) Localization in Cell Plates of Wild-Type and fra2 Given the importance of callose for cell plate establishment and integrity [8,22], its distribution was investigated by immunolocalization. Confocal laser scanning microscope (CLSM) observations revealed a stronger and more widespread signal in meristematic root cells of the wild-type (Figure7a), in comparison with the mutant (Figure7b), which was also confirmed by fluorescence intensity measurements (Figure7c). In particular, callose signal was unexpectedly low or occasionally absent, even in cytokinetic cells with developing cell plates (Figure7b). The distribution of KNOLLE was also followed by CLSM after immunolocalization, since this syntaxin appears pivotal for higher plant cytokinesis [6]. KNOLLE was found in dividing cells of both wild-type and mutant roots, with a prominent signal on cell plates of all cytokinetic cells, nascent cross walls of all post cytokinetic cells, and the transverse cell wall of certain interphase cells (Figure8). However, interphase cells Int. J. Mol. Sci. 2021, 22, 1405 with a visible KNOLLE signal at the transverse cell walls were significantly fewer5 in of the 15 mutant, compared to the wild type (Figure8d).

Figure 3.3. SingleSingle confocalconfocal laser laser scanning scanning microscope microscope (CLSM) (CLSM) sections sections depicting depicting wild-type wild-type ((a), (a, arrows) arrows) and andfra2 fra2(b) cytokinetic(b) cytoki- cellsnetic aftercells tubulinafter tubulin immunostaining. immunostaining. While theWhile expanding the expanding phragmoplast phragmoplast of wild-type of wild-type cell (a) consists cell (a) ofconsists short microtubules, of short microtubules, restricted at the edges of the cell plate, those of fra2 (b, arrowheads) cells are long, bended, and extended to- restricted at the edges of the cell plate, those of fra2 ((b), arrowheads) cells are long, bended, and extended towards the wards the daughter nuclei, exhibiting a ‘‘double-arrow’’ shape. Scale bar 10 μm. daughter nuclei, exhibiting a “double-arrow” shape. Scale bar 10 µm.

InSince fra2 theroots, presence cell plates ofdemethylesteriﬁed of cytokinetic cells exhibited pectins may several be used structural as an defects. indicator Early for cellthe degreeplates of of mid-cytokineti cell plate/crossc wallcells maturationexhibited asymmetrical, [8], demethylesteriﬁed unilateral homogalaturonansgrowth (Figure 4), while(DeSPHGs) in many were cases traced the in phragmoplast cytokinetic and microt post-cytokineticubules extended root cells towards by JIM5 the (anti-DeSPHGdaughter nu- cleiantibody) (Figure immunolocalization. 4b–d, Figure A(1c,d)), In an cells observation with newly in formedaccordance cell plates,with observations JIM5 signal with was tubulinpresent inimmunostaining both fra2 and the (see wild Figure type. 3b). However, In addition, cell plates in several of cytokinetic cases, phragmoplastfra2 root cells were mi- crotubulesmore strongly were labeled observed (Figure to penetrate9b), compared throug toh thosethe whole of wild-type cell plate cells width, at a similar“protruding” stage, intowhere the JIM5 neighboring localization cytoplasm appeared (Figure weaker A(1b)). (Figure In post-cytokinetic9a; cf. Figure9b). cells, In post-cytokinetic cross walls were discontinuous,cells, nascent cross consisting walls wereof still also growing labeled cell by plate JIM5 parts signal (Figure (Figure 5a–b 10).3)However, and unilaterally in fra2 maturedJIM5 distribution cross wall was fragments uneven (Figure (Figure 10 5ab,c;2). cf.In Figuresome cases,10a). the cross walls were abnormally developed, lined by aggregations of multilamellar bodies (Figure 5c–c3), most probably representing remnants of vesicle membranes. Phragmoplasts persisted in post-cytokinetic cells, exhibiting numerous microtubules (Figure 5b–b3). Moreover, post-cytokinetic cell walls exhibited extensive gaps (Figure 6) or persisting phragmoplast microtubules penetrating through the newly-formed cell wall (Figure 6).

Int. J. Mol. Sci. 2021, 22, 1405 6 of 14 Int. J. Mol. Sci. 2021, 22, 1405 6 of 15

Figure 4. TEM micrographsmicrographs ofof aa cytokinetic cytokineticfra2 fra2root root cell cell at at central central longitudinal longitudinal section. section. (a) ( Lowera) Lower magnification magnification view view of the of the whole cell. The daughter nuclei (N) exhibit telophase morphology with condensed chromatin. (b–d) Higher magni- whole cell. The daughter nuclei (N) exhibit telophase morphology with condensed chromatin. (b–d) Higher magnification fication images of the corresponding areas, defined by rectangles in a. Numerous phragmoplast microtubules (arrow- images of the corresponding areas, defined by rectangles in (a). Numerous phragmoplast microtubules (arrowheads) appear heads) appear bending towards the nucleus, some of which extending up to the nuclear surface (b,c). The cell plate (de- b c bendingfined by towardsarrows in the a) nucleus,consists of some numerous of which unaligned extending vesicles up to the (arrows nuclear in surfaceb; compare ( , ).with The the cell aligned plate (defined cell plate by vesicles arrows in (thea)) wild-type consists of in numerous Figure 1). unaligned CW, cell wall. vesicles Bars: (arrows a, 5 μm; in (b–d,b); compare 200 nm. with the aligned cell plate vesicles in the wild-type in Figure1). CW, cell wall. Bars: ( a), 5 µm; (b–d), 200 nm.

Int. J. Mol. Sci. 2021, 22, 1405 7 of 14

Int. J. Mol. Sci. 2021, 22, 1405 7 of 15

Figure 5. TEM micrographs of post-cytokinetic fra2 root cells. (a–c): Lower magnification view of the whole cells. The Figure 5. TEM micrographsdaughter nuclei ofdepicted post-cytokinetic (N) exhibit interphasefra2 morphologyroot cells. (a– (ca).– Higherc): Lower magnification magnification images of the viewcorresponding of the whole cells. The daughter nuclei depictedareas, defined (N) by exhibit rectangles interphase in a–c. (a1–a3): morphology The cell plate/cross (a –wallc). is Higher unevenly magnification consolidated, still not images connected of with the the corresponding areas, parent wall at the right part (a3), where phragmoplast microtubules still persist (a3, arrowheads). At the left part (a1) the defined by rectanglescross inwall, a–c. though (a1 –connecteda3): The to cellthe parent plate/cross wall, is highly wall perforated. is unevenly Close to consolidated, the nucleus (a2) the still cross not wall connected appears with the parent wall at the right partunevenly (a ), thick where and phragmoplastdiscontinuous (a2, arrows), microtubules also baring large still gaps. persist (b1–b (3a): Cell, arrowheads). plate/cross wall is At not the consolidated left part at (a ) the cross wall, the left3 side (b1,b2), though connected with the parent wall at the right (b3). Numerous3 phragmoplast microtubules (b1, b2, 1 though connectedarrowheads) to the parent are still wall, prominent. is highly (c1–c3): The perforated. cross wall is consolidated, Close to the connected nucleus with (thea2 parent) the wall cross at the wall left (c appears1) and unevenly thick right (c3). At its central part, large multilamellar bodies can be observed in touch with the cell plate, while arrows point to and discontinuousmicrotubules (a2, arrows), (c2). CW, also cell wall. baring Bars: largea–c, 5 μm; gaps. a1–c3, 200 (b1 nm.–b 3): Cell plate/cross wall is not consolidated at the left side (b1,b2), though connected with the parent wall at the right (b3). Numerous phragmoplast microtubules (b1, b2, arrowheads) are still prominent. (c1–c3): The cross wall is consolidated, connected with the parent wall at the left (c1) and right (c3). At its central part, large multilamellar bodies can be observed in touch with the cell plate, while arrows point to microtubules Int. J. Mol. Sci. 2021, 22, 1405 8 of 15

(c2). CW, cell wall. Bars: (a–c), 5 µm; (a1–c3), 200 nm.

Figure 6. Light (a) and TEM (b–e) micrographs of post-cytokinetic fra2 root cells. (a) Light micrograph of root cap cells, Figure 6. Light (a)exhibiting and TEM a cell (wallb– e(arrowhead)) micrographs with a larg ofe gap, post-cytokinetic with a vacuole penetratingfra2 root through cells. it. (b,d ()a Lower) Light magnification micrograph view of root cap cells, exhibiting a cell wallof whole (arrowhead) cells. The daughter with nuclei a large (N) gap,exhibit withinterphase a vacuole morphology. penetrating (c) Higher magnification through it.of the (b area,d) Lowerdefined by magnification view rectangle in (b): Cross wall gap (arrows) with a vacuole (V) and mitochondria (mt) almost penetrating through it. (e) of whole cells. TheHigher daughter magnification nuclei of the (N) area exhibit defined by interphase rectangle in (d): morphology. Cross wall gap with (c )microt Higherubules magnification(arrowheads) penetrating of the area defined by rectangle in (b): Crossthrough wall it. CW, gap cell (arrows) wall. Bars: witha, 5 μm; a b, vacuole d, 2 μm, c, (V)e, 200 and nm. mitochondria (mt) almost penetrating through it. (e) Higher magnification of the area defined by rectangle2.3. Callose, in KNOLLE, (d): Cross and Demethylesterified wall gap with Ho microtubulesmogalacturonan (DeSPH (arrowheads)G) Localization penetrating in through it. CW, cell wall. Bars: (a), 5 µm; (b,d), 2Cellµm, Plates (c,e of), Wild-Type 200 nm. and fra2 Given the importance of callose for cell plate establishment and integrity [8,22], its distribution was investigated by immunolocalization. Confocal laser scanning microscope (CLSM) observations revealed a stronger and more widespread signal in meristematic root cells of the wild-type (Figure 7a), in comparison with the mutant (Figure 7b), which was also confirmed by fluorescence intensity measurements (Figure 7c). In particular, callose signal was unexpectedly low or occasionally absent, even in cytokinetic cells with developing cell plates (Figure 7b). The distribution of KNOLLE was also followed by CLSM after immunolocalization, since this syntaxin appears pivotal for higher plant cytokinesis [6]. KNOLLE was found in dividing cells of both wild-type and mutant roots, with a prominent signal on cell plates of all cytokinetic cells, nascent cross walls of all post cytokinetic cells, and the transverse cell wall of certain interphase cells (Figure 8). However, interphase cells with a visible KNOLLE signal at the transverse cell walls were significantly fewer in the mutant, compared to the wild type (Figure 8d). Since the presence of demethylesterified pectins may be used as an indicator for the degree of cell plate/cross wall maturation [8], demethylesterified homogalaturonans

Int. J. Mol. Sci. 2021, 22, 1405 8 of 14

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 16

Figure 7. (a,b) Single CLSM sections depicting callose (anti-β-1,3-glucan) localization (green) in root cells of wild-type (a) and fra2 (b). DNA counterstaining appears in red (pseudocolor). (c) Graph illustrating the corrected total cell ﬂuorescence (CTCF) intensity measurements of callose (anti-β-1,3-glucan) localization in wild-type and fra2. The newly created cell walls of wild-type root cells are intensively stained with the anti-β-1,3-glucan antibody (a), while in fra2 root cytokinetic cells it exhibits a reduced signal (b), or appears occasionally absent (not any callose signal between sister chromosome groups in the white circle). (c) CTCF is statistically signiﬁcantly reduced (asterisk, * p < 0.05) in fra2 root cells. Bar: 10 µm. Int. J. Mol. Sci. 2021, 22, 1405 9 of 14 Int. J. Mol. Sci. 2021, 22, 1405 10 of 15

Int. J. Mol. Sci. 2021, 22, 1405 10 of 15

Figure 8. (a,b) Single CLSM sections depicting anti-KNOLLE (red) and anti-α-tubulin (green) localization in root cells of Figure 8. (a,b) Single CLSM sections depicting anti-KNOLLE (red) and anti-α-tubulin (green) localization in root cells of wild-typewild-type (a) and (afra2) and( bfra2).DNA (b). DNA counterstaining counterstaining appears appears in in blue. blue. ((c) Graph illustrating illustrating the the corrected corrected total total cell fluorescence cell fluorescence Figure 8. (a,b) Single CLSM sections depicting anti-KNOLLE (red) and anti-α-tubulin (green) localization in root cells of (CTCF)(CTCF) intensity intensity measurements measurements of anti-KNOLLE of anti-KNOLLE localization localization in in wild-typewild-type and and fra2.fra2 (.(d)d Graph) Graph illustrating illustrating the cytokinetic, the cytokinetic, wild-typepost-cytokinetic, (a) and fra2 and(b). earlyDNA interphase counterstaining cell percentage appears co inunts blue. exhibiting (c) Graph anti-KNOLLE illustrating signal the corrected in wild type total and cell fra2. fluorescence (a,b): post-cytokinetic, and early interphase cell percentage counts exhibiting anti-KNOLLE signal in wild type and fra2. (a,b): (CTCF)The intensity developing measurements cell plates (arrows of anti in-KNOLLE a, b) in both localization wild-type and in wild-type fra2 and the and newly fra2. developed (d) Graph cell illustrating walls (arrowheads the cytokinetic, in Thepost-cytokinetic, developinga) of wild-type cell andplates root early cells (arrows interphase are intensely in a, cell b) stained in percentage both with wild-type the co anti-KNOLLEunts and exhibitingfra2 and antibody. theanti-KNOLLE newly (c,d) developedCTCF signal of KNOLLE cellin wild walls is typenot (arrowheads statistically and fra2. ( ina,b a)): ofThe wild-type developingsignificantly root cell cellsreduced plates are ( intenselyp(arrows > 0.05) in instained fra2 a, b)root in with cells,both thehowever, wild-type anti-KNOLLE it isand statistically fra2 antibody. and significantly the newly (c,d) CTCF developedreduced of KNOLLE(asterisk cell walls in isd; not p(arrowheads < 0.05) statistically in in significantlya) of wild-typefra2 early reduced interphaseroot cells (* p >arecells. 0.05) intensely Bar: in 10fra2 μ m.stainedroot cells, with however, the anti-KNOLLE it is statistically antibody. significantly (c,d) CTCF reduced of KNOLLE (asterisk is in not (d); statistically * p < 0.05) insignificantlyfra2 early interphase reduced (p cells. > 0.05) Bar: in10 fra2µm. root cells, however, it is statistically significantly reduced (asterisk in d; p < 0.05) in fra2 early interphase cells. Bar: 10 μm.

Figure 9. Single CLSM sections depicting DeSPHG localization by the JIM5 (green) antibody in cytokinetic root cells of wild-type (a) and fra2 (b). DNA counterstaining appears in red (pseudocolor). The developing cell plate (defined by arrowheads) of wild-type root cells is poorly stained with the JIM5 antibody (a), while in fra2 it exhibits a stronger JIM5 signal (b, arrowheads). Bar: 10 μm.

FigureFigure 9. SingleSingle CLSM CLSM sections sections depicting depicting DeSPHG DeSPHG loca localizationlization by by the the JIM5 JIM5 (green) (green) antibody antibody in in cytokinetic root cells of wild-type (a) and fra2 (b). DNA counterstaining appears in red (pseudo- cytokinetic root cells of wild-type (a) and fra2 (b). DNA counterstaining appears in red (pseudocolor). color). The developing cell plate (defined by arrowheads) of wild-type root cells is poorly stained The developing cell plate (deﬁned by arrowheads) of wild-type root cells is poorly stained with the with the JIM5 antibody (a), while in fra2 it exhibits a stronger JIM5 signal (b, arrowheads). Bar: 10 μm. JIM5 antibody (a), while in fra2 it exhibits a stronger JIM5 signal ((b), arrowheads). Bar: 10 µm.

Int. J. Mol. Sci. 2021, 22, 1405 10 of 14 Int. J. Mol. Sci. 2021, 22, 1405 11 of 15

FigureFigure 10.10. DeSPHGDeSPHG localizationlocalization byby thethe JIM5JIM5 (green)(green) antibodyantibody inin post-cytokineticpost-cytokinetic cells of wild-type ((aa)) andand fra2 ((b,,cc)) roots. roots. DNA DNA counterstaining counterstaining appears appears blue. blue. JIM5 JIM5 fluorescence fluorescence reveals reveals that the that con- the solidated cross wall (defined by arrowheads in all figures) is evenly thick in the wild-type (a), while consolidated cross wall (defined by arrowheads in all figures) is evenly thick in the wild-type (a), in fra2 it appears unevenly thick (arrow in b) or with gaps (arrow in c). Bars: 10 μm. while in fra2 it appears unevenly thick (arrow in (b)) or with gaps (arrow in (c)). Bars: 10 µm.

3.3. DiscussionDiscussion AccordingAccording toto our our observations, observations, p60-katanin p60-katanin malfunction malfunction results results in defective in defective cell plate cell formationplate formation and maturation. and maturation. The observed The observed defects defects can be distinguishedcan be distinguished as structural as structural (shape, width,(shape, integrity) width, integrity) and chemical and (polysaccharidechemical (polysaccharide distribution). distribution). Apart from Apart confirming from con- the defectivefirming the phragmoplast defective phragmoplast morphology morphology [19,20] and [19,20] expansion and expansion rate [21], already rate [21], reported already byreported CLSM by studies, CLSM the studies, data presentedthe data presented here offer here a deeper offer a view deeper in theview cytokinetic in the cytokinetic defects commonlydefects commonly found in found p60-katanin in p60-katanin mutants. mutants. DelayedDelayed phragmoplast/cellphragmoplast/cell plate plate expansion [21] [21] may well be the cause forfor thethe dif-dif- ferenceference inin cellcell plateplate contents.contents. ItIt isis well-establishedwell-established thatthat cellcell plateplate vesiclesvesicles initially initially contain contain highlyhighly esterifiedesterified homogalacturonans, homogalacturonans, while while demethylesterified demethylesterified homogalacturonans homogalacturonans appear ap- laterpear duringlater during maturation maturation [8]. [8]. On On the the contrary, contrary, in fra2in fra2cytokinetic cytokinetic cells, cells, cell cell plates plates were were enrichedenriched withwith demethylesterifieddemethylesterified homogalacturonanshomogalacturonans eveneven atat earlyearly cytokinesis,cytokinesis, whichwhich waswas furtherfurther intensified intensified in in post-cytokinetic post-cytokinetic cells. cells. In parallel,In parallel, although although callose callose is universally is univer- presentsally present as a “solidifier” as a “solidifier” in expanding in expanding cell plates cell plates [8], its [8], presence its presence in fra2 incytokinetic fra2 cytokinetic cells wascells significantly was significantly decreased. decreased. Taken Taken together, together, the above the findingsabove findings indicate indicate that, due that, to phrag-due to moplastphragmoplast defects, defects, the formation, the formation, consolidation, consolidation, and expansion and expansion of the cell of platethe cell cannot plate keep can- pacenot keep with pace its maturation with its maturation in dividing infra2 dividingroot cells. fra2Therefore, root cells. asTherefore, a consequence as a consequence of delayed cytokinesisof delayed cytokinesis [21], the ingredients [21], the ingredients of the cell plate of the are cell modified plate are earlier modified than earlier expected, than i.e., ex- duringpected, cell i.e., plate during expansion cell plate than expansion after its than fusion after with its thefusion parent with walls. the parent walls. TheThe aboveabove claimclaim isis furtherfurther supportedsupported byby thethe distribution distributionof of KNOLLE KNOLLE syntaxin. syntaxin. InIn general,general, KNOLLEKNOLLE isis presentpresent duringduring thethe wholewhole processprocess ofof cytokinesis,cytokinesis, alsoalso remainingremaining temporarilytemporarily atat early early interphase interphase in in the the nascent nascent cross cross wall wall [6 ,[6,7].7]. Accordingly, Accordingly, its presenceits presence in newlyin newly formed formed cell walls cell ofwalls interphase of interphase cells, asobserved cells, as in observed wild-type in roots, wild-type is well-expected roots, is (Figurewell-expected8a). On (Figure the contrary, 8a). On in thefra2 contrary,roots, interphase in fra2 roots, cells interphase with a KNOLLE cells with signal a KNOLLE were remarkablysignal were scarce,remarkably in comparison scarce, in tocomparison the wild type. to the This wild difference type. This may difference be attributed may tobe theattributed delayed to cytokinesis the delayed of fra2cytokinesisdividing of cells: fra2 asdividing cytokinesis cells: takes as cytokinesis longer than takes normal longer to bethan completed, normal to the be entrancecompleted, of post-cytokineticthe entrance of post-cytokinetic cells to interphase cells is to also interphase delayed. is As also a consequence,delayed. As a KNOLLE consequence, does notKNOLLE catch up does with not the catch cytokinesis/interphase up with the cytokinesis/interphase transition and is depletedtransition from and newis depleted cell walls from prematurely. new cell walls prematurely. The main structural defects observed in fra2 cell plates and cross walls were the The main structural defects observed in fra2 cell plates and cross walls were the in- increased and uneven thickness, presence of multilamellar bodies and large gaps through creased and uneven thickness, presence of multilamellar bodies and large gaps through the whole cell plate surface. One reason for building an unevenly thick cell plate may be the whole cell plate surface. One reason for building an unevenly thick cell plate may be the increased length of phragmoplast microtubules. De Keijer et al. [9] have shown in the increased length of phragmoplast microtubules. De Keijer et al. [9] have shown in Physcomitrella patens that when phragmoplast microtubules were excessively overlapping Physcomitrella patens that when phragmoplast microtubules were excessively overlapping and their (+) ends penetrated through the expected mid-zone, a thick and irregularly shaped and their (+) ends penetrated through the expected mid-zone, a thick and irregularly cell plate was built. In cytokinetic cells of fra2 root, microtubules were also observed to shaped cell plate was built. In cytokinetic cells of fra2 root, microtubules were also ob- penetrate through the whole cell plate plane (Figures A1 and6e), which may result in served to penetrate through the whole cell plate plane (Figure A1, 6e), which may result some of the malformations observed. In addition, the increased stability and persistence of in some of the malformations observed. In addition, the increased stability and persis-

Int. J. Mol. Sci. 2021, 22, 1405 11 of 14

microtubules in the so-called lagging phragmoplast zone [4] may result in excess vesicle transport and fusion at already consolidated cell plate areas, probably responsible for the formation of multilamellar bodies (Figure5c). Moreover, a mechanical dislocation of cell plate parts, due to “pushing” by elongated phragmoplast microtubules, may account for the failure of those parts to make a proper junction (Figure5a). An explanation why cell plate gaps persist and are sometimes inherited by nascent cell walls (Figure6a–c), may be the early cell plate maturation, in comparison with its expansion, also combined with the lack of the stabilizing effect of callose. Gaps are created, because of phragmoplast defects, then fail to be healed, as cell plate matures and attains the properties of a cross wall (presence of demethylesterified homogalacturonans, low callose content) even before its eventual fusion with the parent walls. In support of such a view, perforated cell plates were prominent in callose-defective massue mutants of A. thaliana [22]. Moreover, the influence of defective preprophase microtubule band organization in p60-katanin mutants should not be overlooked. Preprophase bands in the above mutants are notoriously malformed, asymmetrically organized or even incomplete [19–21]. Conse- quently, division site demarcation and properties are expected to be compromised in fra2 dividing cells. The pivotal role of the division site, pre-established by preprophase band organization, in proper cell plate maturation and flattening have been already reported [23]. It could be thus assumed that due to faulty preprophase band organization, the division site cannot exert its effect on cell plate “finishing” to a new cross wall. In conclusion, phragmoplast defects due to p60-katanin malfunction result in faulty cell plate and cross wall formation during fra2 cytokinesis. In particular, delayed phragmoplast expansion and increased microtubule length lead to a loss of synchronization between the growth and chemical maturation of the cell plate. Consequently, cell plates exhibit structural defects, such as uneven thickness and gaps, which are then bequeathed to the nascent cross walls. A challenge for further research is to investigate the effect of such cytokinetic abnormalities on specialized cell types, especially those occurring by asymmetric divisions, such as meristemoids and trichome initials.

4. Materials and Methods Plant materials, namely seeds of wild type (Col-0) and the fra2 p60-katanin A. thaliana mutant, were purchased from the Nottingham Arabidopsis Stock Center (NASC). All the chemicals and reagents used in this study were supplied by Sigma (Taufkirchen, Germany), Merck (Darmstadt, Germany), and Applichem (Darmstadt, Germany), and all the following steps were carried out at room temperature, unless stated otherwise. Seeds of the wild-type and fra2 were bleach-surface-sterilized and grown on solid 1/2 MS agar medium as previously described [24]. Roots of 5-day-old wild-type and fra2 seedlings were prepared for transmission electron microscopy (TEM) by the protocol in [25]. In brief, root segments from at least 15 roots, comprising the meristematic root zone, were fixed for 4 h in 3% (v/v) glutaraldehyde in 50 mM sodium cacodylate, pH 7, post-fixed in 1% (w/v) osmium tetroxide in the same buffer for 3 h, dehydrated in an acetone series and embedded in Spurr’s resin. Ultrathin sections (70–90 nm), double stained with uranyl acetate and lead citrate, were observed with a JEOL JEM 1011 TEM at 80 kV and micrographs were acquired with a Gatan ES500 W camera. Root segments of both wild- type and fra2 seedlings were also prepared for immunodetection of demethylesterified homogalacturonans (DeSPHGs) with the JIM5 rat antibody (Plant Probes, University of Leeds), while anti-β-1,3-glucan was conducted according to [26]. In short, root tips were fixed in 4% (w/v) paraformaldehyde in PEM buffer (50 mM PIPES, 5 mM EGTA, 5 mM MgSO4, pH 6.8) for 1 h. Fixed roots were rinsed twice in the same buffer for 10 min. Cell walls were digested for 1 h in 2% (w/v) Cellulase Onozuka R-10 (Duchefa, Haarlem, Netherlands) in PEM. Then, root tips were extracted with 5% (v/v) DMSO + 1% (v/v) Triton X-100 in PBS for 1 h. Incubations with JIM5, anti-β-1,3-glucan and FITC- anti-rat (Invitrogen, Carlsbad, CA), all diluted 1:40 in PBS, were carried out sequentially overnight in the dark with a washing intermediate step (3 × 10 min). Finally, after DNA Int. J. Mol. Sci. 2021, 22, 1405 12 of 14

counterstaining with DAPI and washing in PBS, the specimens were mounted in a PBS- glycerol mixture (1:2 v/v), supplemented with 0.5% (w/v) p-phenylenediamine as anti-fade agent, while in some cases the roots were gently squashed between the microscope slide and coverslip, to release some meristematic root cells from the surrounding tissues. α- tubulin and KNOLLE immunolabeling (anti-KNOLLE diluted 1:2000) was conducted as stated in [19]. The specimens were examined with a Zeiss LSM780 confocal laser scanning microscope (CLSM) as previously described [25] or with a Zeiss Axioplan microscope (Zeiss, Berlin, Germany), equipped with a Zeiss Axiocam MRc5 digital camera, using the ZEN 2.0 software, according to the manufacturer‘s instructions. Confocal, epi-fluorescence, and TEM images were processed with Adobe Photoshop with only linear settings. The corrected total cell fluorescence (CTCF) intensity measurements were performed using the Image J (http://rsbweb.nih.gov/ij/) software according to [27]. For CTCF measurements for either callose or anti-KNOLLE immunostaining images, seven roots of wild type and seven of fra2 were accessed and results were obtained from a total number of 1000 cells in each case. Cell counts exhibiting anti-KNOLLE signal in wild-type and fra2 were expressed as percentage. Statistical analyses (ANOVA with Dunnett’s multiple comparison test) were performed using GraphPad software (San Diego, CA, USA), with significance at p < 0.05.

Author Contributions: Conceptualization, E.P. and I.-D.S.A.; methodology, A.K. and D.P.; investigation, E.P., A.K., and I.-D.S.A.; resources, E.P. and I.-D.S.A.; data curation, A.K., D.P., and I.-D.S.A.; writing—original draft preparation, E.P. and I.-D.S.A.; writing—review and editing, E.P., D.P., and I.- D.S.A.; supervision, E.P. All authors have read and agreed to the published version of the manuscript. Funding: E.P. was funded by the AUTh Research Committee, grant number 91913. I.-D.S.A. was funded by the National and Kapodistrian University of Athens. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The anti-KNOLLE antibody was a generous gift of George Komis, Palacky University, Olomouc, Czechia. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

CLSM Confocal laser scanning microscope DeSHGs Demethylesteriﬁed homogalacturonans TEM Transmission electron microscope MAPs Microtubule associated proteins Int. J. Mol. Sci. 2021, 22, 1405 13 of 14 Int. J. Mol. Sci. 2021, 22, 1405 14 of 15

Appendix A Appendix A

Figure A1. SelectedSelected TEM TEM micrographs micrographs of of cytokinetic cytokinetic fra2fra2 rootroot cells, cells, exhibiting exhibiting a variety a variety of deviations of deviations in microtubule in microtubule or- organization.ganization. Phragmoplast Phragmoplast microtubules microtubules are are pointed pointed by by yellow yellow a arrowheads,rrowheads, while while cell cell plates plates ar aree defined defined by red arrows in all micrographs. (a) Phragmoplast microtubules at the expanding edge of the cell plate are bended, forming an acute an- all micrographs. (a) Phragmoplast microtubules at the expanding edge of the cell plate are bended, forming an acute angle gle with it. Some of them appear branched (branch point inside the white circle). (b) A microtubule penetrating through with it. Some of them appear branched (branch point inside the white circle). (b) A microtubule penetrating through the the whole cell plate width can be observed at the right part (beside the right arrow) (c,d) Phragmoplast microtubules ex- wholetend from cell the plate cell width plate can rim be to observedthe nuclear at the(N) rightsurface part (see (beside also Figure the right 4). arrow)Bars: 500 (c ,nm.d) Phragmoplast microtubules extend from the cell plate rim to the nuclear (N) surface (see also Figure4). Bars: 500 nm. References References 1. Buschmann, H.; Müller, S. Update on plant cytokinesis: Rule and divide. Curr. Opin. Plant Biol. 2019, 52, 97–105. Curr. Opin. Plant Biol. 2019 52 1.2. Buschmann,Livanos, P.; Müller, H.; Müller, S. Division S. Update plane on establishment plant cytokinesis: and cytokinesis. Rule and divide. Annu. Rev. Plant Biol. 2019, 70, 239–267., , 97–105. [CrossRef] 3. [Smertenko,PubMed] A.; Assaad, F.; Bezanilla, M.; Buschmann, H.; Drakakaki, G.; Hauser, M.; Janson, M.; Mineyuki, Y.; Moore, I.; Annu. Rev. Plant Biol. 2019 70 2. Livanos,Müller, S.; P.; et Müller, al. Plant S. Divisioncytokinesis: plane Terminology establishment for and structures cytokinesis. and processes. Trends Cell Biol. ,2017, 239–267., 27, 885–894. [CrossRef ][PubMed] 3. Smertenko, A.; Assaad, F.; Bezanilla, M.; Buschmann, H.; Drakakaki, G.; Hauser, M.; Janson, M.; Mineyuki, Y.; Moore, I.; Müller, 4. Smertenko, A. Phragmoplast expansion: The four-stroke engine that powers plant cytokinesis. Curr. Opin. Plant Biol. 2018, 46, S.; et al. Plant cytokinesis: Terminology for structures and processes. Trends Cell Biol. 2017, 27, 885–894. [CrossRef][PubMed] 130–137. 4. Smertenko, A. Phragmoplast expansion: The four-stroke engine that powers plant cytokinesis. Curr. Opin. Plant Biol. 2018, 46, 5. Smertenko, A.; Hewitt, S.L.; Jacques, C.N.; Kacprzyk, R.; Liu, Y.; Marcec, M.J.; Moyo, L.; Ogden, A.; Oung, H.M.; Schmidt, S.; et 130–137. [CrossRef][PubMed] al. Phragmoplast microtubule dynamics—A game of zones. J. Cell Sci. 2018, 131, jcs203331. 5. Smertenko, A.; Hewitt, S.L.; Jacques, C.N.; Kacprzyk, R.; Liu, Y.; Marcec, M.J.; Moyo, L.; Ogden, A.; Oung, H.M.; Schmidt, S.; et al. 6. Lauber, M.H.; Waizenegger, I.; Steinmann, T.; Schwarz, H.; Mayer, U.; Hwang, I.; Lukowitz, W.; Jürgens, G. The Arabidopsis Phragmoplast microtubule dynamics—A game of zones. J. Cell Sci. 2018, 131, jcs203331. [CrossRef] KNOLLE protein is a cytokinesis-specific syntaxin. J. Cell Biol. 1997, 139, 1485–1493. 6. Lauber, M.H.; Waizenegger, I.; Steinmann, T.; Schwarz, H.; Mayer, U.; Hwang, I.; Lukowitz, W.; Jürgens, G. The Arabidopsis 7. Touihri, S.; Knöll, C.; Stierhof, Y.-D.; Müller, I.; Mayer, U.; Jürgens, G. Functional anatomy of the Arabidopsis cytokine- KNOLLE protein is a cytokinesis-specific syntaxin. J. Cell Biol. 1997, 139, 1485–1493. [CrossRef] sis-specific syntaxin KNOLLE. Plant J. 2011, 68, 755–764. 7. Touihri, S.; Knöll, C.; Stierhof, Y.-D.; Müller, I.; Mayer, U.; Jürgens, G. Functional anatomy of the Arabidopsis cytokinesis-specific 8. Drakakaki, G. Polysaccharide deposition during cytokinesis: Challenges and future perspectives. Plant Sci. 2015, 236, 177–184. syntaxin KNOLLE. Plant J. 2011, 68, 755–764. [CrossRef] 9. De Keijzer, J.; Kieft, H.; Ketelaar, T.; Goshima, G.; Janson, M.E. Shortening of microtubule overlap regions defines membrane delivery sites during plant cytokinesis. Curr. Biol. 2017, 27, 514–520. 10. Hamada, T. Microtubule-associated proteins in higher plants. J. Plant Res. 2007, 120, 79–98.

Int. J. Mol. Sci. 2021, 22, 1405 14 of 14

8. Drakakaki, G. Polysaccharide deposition during cytokinesis: Challenges and future perspectives. Plant Sci. 2015, 236, 177–184. [CrossRef] 9. De Keijzer, J.; Kieft, H.; Ketelaar, T.; Goshima, G.; Janson, M.E. Shortening of microtubule overlap regions defines membrane delivery sites during plant cytokinesis. Curr. Biol. 2017, 27, 514–520. [CrossRef] 10. Hamada, T. Microtubule-associated proteins in higher plants. J. Plant Res. 2007, 120, 79–98. [CrossRef] 11. Nakamura, M.; Ehrhardt, D.W.; Hashimoto, T. Microtubule and katanin-dependent dynamics of microtubule nucleation com- plexes in the acentrosomal Arabidopsis cortical array. Nat. Cell Biol. 2010, 12, 1064–1070. [CrossRef][PubMed] 12. Bichet, A.; Desnos, T.; Turner, S.; Grandjean, O.; Höfte, H. BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis. Plant J. 2001, 25, 137–148. [CrossRef][PubMed] 13. Burk, D.H.; Liu, B.; Zhong, R.; Morrison, W.H.; Ye, Z.H. A katanin-like protein regulates normal cell wall biosynthesis and cell elongation. Plant Cell 2001, 13, 807–827. [CrossRef] 14. Webb, M.; Jouannic, S.; Foreman, J.; Linstead, P.; Dolan, L. Cell specification in the Arabidopsis root epidermis requires the activity of ECTOPIC ROOT HAIR 3—A katanin-p60 protein. Development 2002, 129, 123–131. 15. Bouquin, T.; Mattsson, O.; Næsted, H.; Foster, R.; Mundy, J. The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth. J. Cell Sci. 2003, 116, 791–801. [CrossRef] 16. Luptovˇciak,I.; Komis, G.; Takáˇc,T.; Oveˇcka,M.; Šamaj, J. Katanin: A sword cutting microtubules for cellular, developmental, and physiological purposes. Front. Plant Sci. 2017, 8, 1982. [CrossRef] 17. Luptovˇciak,I.; Samakovli, D.; Komis, G.; Šamaj, J. KATANIN 1 is essential for embryogenesis and seed formation in Arabidopsis. Front. Plant Sci. 2017, 8, 728. [CrossRef] 18. Oveˇcka,M.; Luptovˇciak,I.; Komis, G.; Šamajová, O.; Samakovli, D.; Šamaj, J. Spatiotemporal pattern of ectopic cell divisions contribute to mis-shaped phenotype of primary and lateral roots of katanin1 mutant. Front. Plant Sci. 2020, 11, 734. [CrossRef] 19. Panteris, E.; Adamakis, I.-D.S.; Voulgari, G.; Papadopoulou, G. A role for katanin in plant cell division: Microtubule organization in dividing root cells of fra2 and lue1 Arabidopsis thaliana mutants. Cytoskeleton 2011, 68, 401–413. [CrossRef] 20. Panteris, E.; Adamakis, I.-D.S. Aberrant microtubule organization in dividing root cells of p60-katanin mutants. Plant Signal. Behav. 2012, 7, 16–18. [CrossRef] 21. Komis, G.; Luptovˇciak,I.; Oveˇcka,M.; Samakovli, D.; Šamajová, O.; Šamaj, J. Katanin effects on dynamics of cortical microtubules and mitotic arrays in Arabidopsis thaliana revealed by advanced live-cell imaging. Front. Plant Sci. 2017, 8, 866. [CrossRef] [PubMed] 22. Thiele, K.; Wanner, G.; Kindzierski, V.; Jürgens, G.; Mayer, U.; Pachl, F.; Assad, F.F. The timely deposition of callose is essential for cytokinesis in Arabidopsis. Plant J. 2009, 58, 13–26. [CrossRef][PubMed] 23. Mineyuki, Y.; Gunning, B.E.S. A role for preprophase bands of microtubules in maturation of new cell walls, and a general proposal on the function of preprophase band sites in cell division in higher plants. J. Cell Sci. 1990, 97, 527–537. 24. Adamakis, I.D.S.; Panteris, E.; Eleftheriou, E.P. Tungsten disrupts root growth in Arabidopsis thaliana by PIN targeting. J. Plant Physiol. 2014, 171, 1174–1187. [CrossRef][PubMed] 25. Panteris, E.; Diannelidis, B.-E.; Adamakis, I.-D.S. Cortical microtubule orientation in Arabidopsis thaliana root meristematic zone depends on cell division and requires severing by katanin. J. Biol. Res. 2018, 25, 12. [CrossRef][PubMed] 26. Meidani, C.; Ntalli, N.G.; Giannoutsou, E.; Adamakis, I.-D.S. Cell wall modifications in giant cells induced by the plant parasitic nematode Meloidogyne incognita in wild-type (Col-0) and the fra2 Arabidopsis thaliana katanin mutant. Int. J. Mol. Sci. 2019, 20, 5465. [CrossRef] 27. Adamakis, I.-D.S.; Panteris, E.; Eleftheriou, E.P. The nitrate reductase inhibitor, tungsten, disrupts actin microfilaments in Zea mays L. Protoplasma 2014, 251, 567–574. [CrossRef]