Two Arabidopsis Phragmoplast-Associated Kinesins Play aCritical Role in Cytokinesis during Male Gametogenesis

Yuh-Ru Julie Lee,a Yan Li,b and Bo Liua,1

a Section of Plant Biology, University of California, Davis, California 95616b State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University,

Beijing 100094, China

In plant cells, cytokinesis is brought about by the phragmoplast. The phragmoplast has a dynamic microtubule array of two

mirrored sets of microtubules, which are aligned perpendicularly to the division plane with their plus ends located at the

division site. It is not well understood how the phragmoplast microtubule array is organized. In Arabidopsis thaliana, two

homologous microtubule motor kinesins, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, localize exclusively at the

juxtaposing plus ends of the antiparallel microtubules in the middle region of the phragmoplast. When either kinesin was

knocked out by T-DNA insertions, mutant plants did not show a noticeable defect. However, in the absence of both kinesins,

postmeiotic development of the male gametophyte was severely inhibited. In dividing microspores of the double mutant,

microtubules often became disorganized following chromatid segregation and failed to form an antiparallel microtubule

array between reforming nuclei. Consequently, the first postmeiotic cytokinesis was abolished without the formation of a

cell plate, which led to failures in the birth of the generative cell and, subsequently, the sperm. Thus, our results indicate that

Kinesin-12A and Kinesin-12B jointly play a critical role in the organization of phragmoplast microtubules during cytokinesis

in the microspore that is essential for cell plate formation. Furthermore, we conclude that Kinesin-12 members serve as

dynamic linkers of the plus ends of antiparallel microtubules in the phragmoplast.

INTRODUCTION

In angiosperms, cytokinesis is brought about by the phragmo-

plast, an apparatus containing a framework of microtubules, to-

gether with actin microfilaments and membranous organelles

(Staehelin and Hepler, 1996). In the phragmoplast, microtubules

are organized in two mirrored sets: their plus ends are juxta-

posed at the division site and their minus ends face the reforming

nuclei. These antiparallel microtubules serve as tracks along

which Golgi-derived vesicles are transported toward microtu-

bule plus ends. Vesicle fusion gives rise to the cell plate, the

physical barrier dividing the cytoplasm of the mother cell. Thus,

the arrangement of this antiparallel array and the positional main-

tenance of microtubule plus ends allow vesicles to be unidirec-

tionally delivered toward microtubule plus ends, to the division

site.

Phragmoplast microtubules are derived from those of the

spindle midzone and are highly dynamic (Zhang et al., 1990). The

establishment of the antiparallel phragmoplast array involves

microtubule–microtubule sliding driven by motors in the Kinesin-5

family (Asada et al., 1997). After the antiparallel pattern is estab-

lished, however, tubulin dimers are continuously polymerized

onto the plus end of the phragmoplast microtubules (Vantard

et al., 1990; Asada et al., 1991). Newly added microtubule seg-

ments at the plus ends would tend to overlap in an antiparallel

fashion. But a recent tomographic investigation of samples

prepared by rapid freezing and freeze substitution indicates

that microtubule plus ends do not overlap in the middle of the

phragmoplast in meristematic cells (Austin et al., 2005). Thus, the

possibility is excluded for microtubules from opposite sets to

slide against each other once the phragmoplast microtubule

array is established. Newly added microtubule segments, there-

fore, have to be continuously translocated away from the middle

line of the phragmoplast in order to have the plus ends remain at

the division site. A plausible scenario is that one or more plus

end–directed kinesins may act at the plus ends of oppositely

oriented phragmoplast microtubules to continuously generate

the outward force.

In the model plant Arabidopsis thaliana, there are 61 genes

encoding microtubule-based motor kinesins in >10 subfamilies

(Reddy and Day, 2001). Among them, two homologous genes,

PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, exhibit a cell

cycle–dependent localization pattern and specifically decorate

the plus ends of phragmoplast microtubules (Lee and Liu, 2000,

2004; Pan et al., 2004). Kinesin-12 members in both plants and

animals bear a signature neck sequence of plus end–directed

kinesins (Lee and Liu, 2000; Miki et al., 2005). Indeed, the

Xenopus Kinesin-12 Xl KLP2 exhibits plus end–directed motility

in vitro (Boleti et al., 1996). It has been hypothesized that Kinesin-

12s serve as dynamic linkers between two mirrored sets of

microtubules in the phragmoplast (Lee and Liu, 2000; Liu and

Lee, 2001).

In this report, we used mutants in which genes encoding

PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B were inacti-

vated by T-DNA insertions for functional studies of these two

1 Address correspondence to bliu@ucdavis.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Bo Liu (bliu@ucdavis.edu).www.plantcell.org/cgi/doi/10.1105/tpc.107.050716

This article is published in The Plant Cell Online, The Plant Cell Preview Section, which publishes manuscripts accepted for publication after they

have been edited and the authors have corrected proofs, but before the final, complete issue is published online. Early posting of articles reduces

normal time to publication by several weeks.

The Plant Cell Preview, www.aspb.org ª 2007 American Society of Plant Biologists 1 of 11

kinesins. The data presented here indicate that Kinesin-12s play

a critical role in organizing phragmoplast microtubules and, con-

sequently, in cytokinesis.

The function of Kinesin-12A/B seemed to be different from

those of reported proteins acting on phragmoplast microtubules.

For example, TKRP125 of the BIMC/Kinesin-5 subfamily func-

tions in the sliding of interdigitated microtubules (Asada et al.,

1997). The At MAP65-3/PLE protein in the MAP65/PRC/Ase1p

family plays a critical role in phragmoplast microtubule organi-

zation by maintaining the dimension of the microtubule array, and

mutations at the corresponding locus lead to the formation of

multinucleated root cells (Muller et al., 2004). Conversely, mi-

crotubule bundling activity conferred by MAP65-1 has to be

downregulated via its phosphorylation by a mitogen-activated

protein kinase cascade in the phragmoplast (Sasabe et al.,

2006). Phragmoplast localization of the kinase is dependent on

two novel plant kinesins that are essential for cytokinesis

(Nishihama et al., 2002; Strompen et al., 2002). Two homologous

kinesins, POK1 and POK2, play a role in the orientation of the

phragmoplast and, consequently, in the orientation of the cell

plate, but not in the organization of phragmoplast microtubules

(Muller et al., 2006). Interestingly, loss of function of these

proteins does not alter the overall organization of the antiparallel

pattern of phragmoplast microtubules. This study indicates that

PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B play a funda-

mental role in the organization of the phragmoplast microtubule

array.

RESULTS

Loss-of-Function Mutations at the Kinesin-12A/B Loci

In order to test whether Kinesin-12A/B are indeed critical for the

organization of phragmoplast microtubules, T-DNA insertional

mutations were recovered (Figure 1A). Mutations at either the

Kinesin-12A or Kinesin-12B locus do not cause a noticeable

defect in the growth and reproduction of the plant (Pan et al.,

2004). We reasoned that these genes might function redundantly

during cytokinesis. Homozygous double mutants were gener-

ated using different alleles of T-DNA insertional mutations (Figure

1A). The kinesin-12a-1 and kinesin-12a-2 mutations had the

T-DNA sequence inserted in the 2nd and 12th exons, respec-

tively, and kinesin-12b-1 and kinesin-12b-2 had T-DNA inserted

in the 1st and 15th exons, respectively. In contrast with single

mutants, the kinesin-12a-1 kinesin-12b-2 and kinesin-12a-2

kinesin-12b-2 double mutants consistently produced signifi-

cantly fewer seeds in their fruits. The result shown in Figure 1B,

as well as other results shown here, were from the kinesin-12a-1

kinesin-12b-2 mutant, unless noted otherwise. Mature siliques of

wild-type plants had ;80% mature seeds. Mature siliques of the

double mutants, however, had <40%. The mutant siliques often

contained small white structures, which could be either unfertil-

ized ovules or fertilized ovules containing early aborted embryos.

While the double mutants produced ;50% fewer seeds than the

wild type, the single mutants produced seeds comparable to

those of wild-type plants (Figure 1C). Because the F1 plants

resembled their homozygous mutant parent (Figure 1C), we

concluded that the incomplete penetrance of the low-fertility

phenotype was inheritable.

Mutations in the Kinesin-12A/B Genes Resulted in Defective

Pollen Grains

Because Arabidopsis is a self-pollinating plant, reduction of seed

formation could be due to defects in either the male or female

Figure 1. Mutations at the Kinesin-12A and Kinesin-12B Loci.

(A) Diagrammatical representation of the Kinesin-12A and Kinesin-12B

gene structures. Introns are shown as lines, and exons are shown as

boxes. Positions of the T-DNA mutational insertions of kinesin-12a-1,

kinesin-12a-2, kinesin-12b-1, and kinesin-12b-2 are shown at top of the

diagrams.

(B) The homozygous double mutant of kinesin-12a-1 and kinesin-12b-2

produced significantly fewer seeds in siliques. Red arrows point to ovule

positions where no seeds were found.

(C) Quantification of seed production in wild-type and mutant siliques. The

y axis represents the percentage of ovule positions with seeds. Plants

bearing homozygous single mutations at either locus produced similar

numbers of seeds as their wild-type counterparts. The homozygous

double mutant and its F1 progeny produced ;50% fewer seeds. Geno-

types of the plants are as follows: 12A 12B for plants with wild-type alleles

at both Kinesin-12A and Kinesin-12B loci; 12a 12B for plants with a

homozygous mutation at the Kinesin-12A locus; 12A 12b for plants with a

homozygous mutation at the Kinesin-12B locus; 12a 12b for the homozy-

gous double mutant; and 12a 12b (F1) for the F1 progeny of 12a 12b.

2 of 11 The Plant Cell

gametophyte, or both, or to defects in embryogenesis. At first,

prefertilization and postfertilization ovules of the mutant were

cleared for microscopic examination using Normaski optics. No

observable defect was detected in either the developing embryo

sacs or developing embryos (data not shown). In order to reveal

whether gametophytes were responsible for the phenotype, we

then performed reciprocal pollination experiments by applying

mutant pollen grains onto wild-type stigma, and vice versa. Our

results indicated that pollen grains of double mutant plants

caused reductions of seed formation in wild-type siliques (aver-

age seed number/silique ¼ 7; n ¼ 7) and wild-type pollen grains

restored the seed production level in mutant siliques (average

seed number/silique ¼ 43; n ¼ 7).

Furthermore, reciprocal crosses were performed to analyze

genetic transmission via gametophytes. When pollen grains of

the Kinesin-12A/kinesin-12a-1; kinesin-12b-2/kinesin-12b-2 or

kinesin-12a-1/kinesin-12a-1; Kinesin-12B/kinesin-12b-2 mutant

were used to pollinate the stigma of a wild-type female parent,

transmission of the double mutant allele (kinesin-12a-1; kinesin-

12b-2) was severely reduced (male transmission efficiency ¼12.9 and 20.7%, respectively) (Table 1). When these mutants

were used as female receivers of the wild-type pollen grains,

transmission of the double mutant allele through the female gam-

ete was moderately reduced (female transmission efficiency ¼64.3 and 61.7%, respectively) (Table 1). Thus, in the double mu-

tants, the phenotype of reduction of seed formation was mainly

caused by defective pollen grains. Because the transmission

through the female gamete was not perfect, it was also not ruled

out that these kinesins might play a role in multiple phragmo-

plasts associated with the cellularization of the female gameto-

phyte.

We further analyzed male gametophytes of the double mutant

by fluorescence and electron microscopy. The pollen grain is a

young male gametophyte in angiosperms. The mature male

gametophyte of flowering plants is composed of three haploid

cells, of which two sperm cells are suspended in the cytoplasm of

the vegetative (pollen tube) cell (McCormick, 2004). Pollen grains

collected from open flowers of both wild-type and double mutant

plants were then examined by staining with the DNA-specific dye

49,6-diamidino-2-phenylindole (DAPI). When wild-type flowers

were open, pollen grains were already mature, and they had two

brightly stained sperm nuclei and a faintly stained vegetative

nucleus (Figure 2A, a and b). Pollen grains isolated from mutant

flowers often contained two loosely packed DNA masses (Figure

2B, a and b), and they resembled the DNA mass of the vegetative

nucleus in wild-type pollen grains. To test whether the abnormal

mutant pollen grains produced a generative cell or sperm, we

examined them by transmission electron microscopy. In wild-

type pollen grains, the vegetative nucleus and sperm (only one

revealed in this section) were found in the pollen cytoplasm

(Figure 2A, c). The sperm nucleus and cytoplasm were com-

pletely isolated from the vegetative cytoplasm by the sperm cell

wall (Figure 2A, d). Defective mutant pollen grains, however,

lacked sperm. Instead, two identical nuclei were found in single

sections (Figure 2B, c). The two nuclei were suspended in the

vegetative cytoplasm, and no cell wall–like structure was de-

tected between them (Figure 2B, d).

While many defective pollen grains were consistently detected

in the double mutants, very few such pollen grains were ob-

served in the wild type and single mutants. The difference among

pollen grains of these different genetic backgrounds was striking

when they were classified into three categories: those with two

sperm nuclei and one vegetative nucleus (2þ1); those with two

similar nuclei (1þ1) or those with one large DNA mass (1); and

those aborted ones with a shrunken appearance (s) (Figure 2C).

In the pollen grains with one large DNA mass, the DNA mass

likely resulted from the overlap of two indiscernible nuclei by

fluorescence microscopy, as sister chromatids segregated nor-

mally in the mutants (data not shown). Alternatively, it was also

not ruled out that two nuclei might fuse to become one. Never-

theless, both scenarios reflected the failure of spermatogenesis.

Ninety-seven percent of wild-type pollen grains contained three

nuclei, 3% contained one or two nuclei, and 0% were aborted

(n ¼ 103). In the kinesin-12a-1 single mutant, the distribution of

pollen grains in these three categories was 96, 3, and 1%,

respectively (n ¼ 529). The homozygous kinesin-12b-1 mutant

had very similar distribution of different pollen grains, as did the

heterozygous Kinesin-12A/kinesin-12a-1; Kinesin-12B/kinesin-

12b-2 mutant. In mutants bearing one copy of either Kinesin-12A

or Kinesin-12B, defective pollen grains were detected more

frequently than in the aforementioned mutants (Figure 2C). In the

kinesin-12a-1 kinesin-12b-2 homozygous double mutant, how-

ever, only 42% of pollen grains contained three nuclei; 41% of

pollen grains were either binucleate or uninucleate, and 17% of

Table 1. Transmission Efficiency of kinesin-12a (12a) and kinesin-12b (12b) Mutations in Reciprocal Crosses between Mutant and Wild-Type Plants

Progeny GenotypeTransmission Efficiency

of 12a; 12b GametesaFemale Parent Male Parent 12A/12a; 12B/12b 12A/12A; 12B/12b 12A/12a; 12B/12B Pb

12A/12A; 12B/12B 12A/12a; 12b/12b 12 93 – _ TE ¼ 12.9% <0.0001

12A/12A; 12B/12B 12a/12a; 12B/12b 18 – 87 _ TE ¼ 20.7% <0.0001

12A/12a; 12b/12b 12A/12A; 12B/12B 45 70 – \ TE ¼ 64.3% 0.02

12a/12a; 12B/12b 12A/12A; 12B/12B 37 – 60 \ TE ¼ 61.7% 0.02

a Calculated transmission efficiency (TE) of 12a;12b double mutant gametes through the male (_ TE) and the female (\ TE) parents. _ TE ¼ ratio of 12A/

12a; 12B/12b and 12A/12A; 12B/12b or that of 12A/12a; 12B/12b and 12A/12a; 12B/12B. \ TE ¼ ratio of 12A/12a; 12B/12b and 12A/12A; 12B/12b or

that of 12A/12a; 12B/12b and 12A/12a; 12B/12B.b P values were calculated by the x2 test based on the expected value of 100% or a 1:1 segregation ratio.

Phragmoplast Kinesins in Plant Cytokinesis 3 of 11

pollen grains were shrunken (n ¼ 1825). These data, therefore,

suggested that the increased loss of functional Kinesin-12A/B

genes was accompanied by the increase of defective pollen

grains. It was noticed that mutants bearing one functional copy of

either Kinesin-12A or Kinesin-12B produced significantly more

than half of normal pollen grains, which could result from pos-

sible inheritance of the wild-type product through meiosis. Thus,

our data suggested that in the double mutant, the failure of

gametogenesis frequently took place due to the failure in cyto-

kinesis, resulting in the absence of the male gamete sperm.

Pollen grains isolated from open flowers of the homozygous

double mutant were subject to germination in vitro. It was found

that defective pollen grains with two similar nuclei were able to

produce pollen tubes as trinucleate pollen grains (data not

shown). Thus, a defective pollen grain and resulting tube would

fail to reach the ovule, which would reduce the fertility in the

double mutant.

Kinesin-12A/B Play a Role in the Organization of

Phragmoplast Microtubules

It was hoped that the T-DNA insertions would inactivate Kinesin-12

gene expression in homozygous mutants. The Kinesin-12A and/

or Kinesin-12B transcripts were detected in the wild type (12A/

12A; 12B/12B) by RT-PCR (Figure 3A). Moreover, a correspond-

ing transcript was detected in mutants bearing either one or two

copies of the wild-type Kinesin-12A or Kinesin-12B gene (12a/

12a; 12B/12B, 12A/12A; 12b/12b, 12a/12a; 12B/12b, and 12A/

12a; 12b/12b). While the At1g13320 transcript encoding protein

phosphatase 2A, as a positive control, was detected in the wild

type and all mutants, corresponding transcripts were not de-

tected in homozygous mutants (Figure 3A). Thus, the insertions

inactivated the expression of the Kinesin-12A/B genes.

To determine the activity of Kinesin-12A/B in the dividing

microspores, antibodies raised against the C-terminal domain of

Kinesin-12A were used. The antibodies recognize both Kinesin-12A

Figure 2. The Double Mutant Failed to Produce Male Gametes.

(A) In the wild type (a), a mature pollen grain contains two sperm and one

vegetative nucleus. The sperm nuclei (arrows) and the vegetative nucleus

(arrowhead) were revealed by DAPI staining. In (b), a differential inter-

ference contrast image shows the pollen appearance. Transmission

electron microscopy images ([c] and [d]) show the vegetative nucleus

(VN) and one sperm cell (arrow) in the pollen cytoplasm. Note that the

sperm cytoplasm was physically separated from the pollen cytoplasm by

a barrier (arrows in [d]). The other sperm cell was not included in this

section. Bars ¼ 10 mm in (b) for (a) and (b), 4 mm in (c), and 1 mm in (d).

(B) In the double mutant, a defective pollen grain failed to produce

sperm. DAPI staining (a) showed two loosely packed DNA masses

(arrowheads) resembling the vegetative nucleus. A differential interfer-

ence contrast image of this pollen is shown in (b). Transmission electron

microscopy images ([c] and [d]) show two similar nuclei (N) suspended in

the pollen cytoplasm. Note that between the nuclear envelopes of the

two nuclei (arrowheads), there was no barrier as seen in ([A], [d]). Bars ¼10 mm in (b) for (a) and (b), 4 mm in (c), and 1 mm in (d).

(C) Quantification of pollen grains in the categories of two sperm nuclei

plus one vegetative nucleus (2þ1), two identical nuclei (1þ1) or one DNA

mass (1), and shrunken appearance (s). The y axis represents the

proportion of pollen grains in each category. Pollen grains in the three

categories were quantified in the wild type (12A/12A; 12B/12B), single

mutants (12a/12a; 12B/12B and 12A/12A; 12b/12b), various heterozy-

gous double mutants (12A/12a; 12B/12b, 12a/12a; 12B/12b, and 12A/

12a; 12b/12b), and the homozygous double mutant (12a/12a; 12b/12b).

4 of 11 The Plant Cell

and Kinesin-12B (Pan et al., 2004). In the wild-type microspores,

Kinesin-12A/B appeared as discrete signals between the form-

ing vegetative nucleus and the generative nucleus (Figure 3B).

No detectable signal was revealed in double mutant microspores

at identical stages, indicating that Kinesin-12A/B were absent

from the phragmoplast in these mutant cells (Figure 3B).

To further elucidate what had caused the frequent failure of cell

division, antitubulin immunofluorescence was performed in mi-

crospores of both the wild type and the double mutant. In wild-

type microspores, upon the completion of mitosis, microtubules

were gradually organized into the typical phragmoplast array

between two reforming daughter nuclei located toward one end

of the microspore (Figure 4A, a to c). At first, microtubules were

organized into an antiparallel array, and a clear dark line was

revealed in the middle (Figure 4A, a). Such a dark line marks the

plus end of phragmoplast microtubules. The progression of

cytokinesis was accompanied by the expansion of the phrag-

moplast microtubule array toward the periphery and the short-

ening of microtubules at their ends (Figure 4A, b). Once the cell

plate was laid down centrifugally, the phragmoplast microtubule

array appeared in a hollow cylinder-like configuration (Figure 4A,

c). The cell plate eventually met the plasma membrane of the

microspore to render a convex lens–shaped generative cell and a

larger vegetative cell (McCormick, 2004).

In dividing microspores of the double mutant, however, mi-

crotubules were frequently disorganized after mitosis was com-

plete (Figure 4B, a to e). In all microspores upon completion of

anaphase, coalesced microtubule bundles were found between

the two sister chromatid masses (Figure 4B, a). At later stages,

microtubule bundles remained unshortened, and their distal

ends toward the reforming daughter (son) nuclei extended at or

near the nuclear envelope (Figure 4B, b and c). The antitubulin

immunofluorescence often did not reveal a dark line in the center

of the microtubule mass (Figure 4B, c). This result suggested that

these microtubules had not had their plus ends organized in the

middle of the phragmoplast, as was seen in the wild-type cells. In

pollen grains collected from open flowers of the homozygous

double mutant, microtubule bundles were still detected, and they

were associated with the nucleus toward the periphery of the

cytoplasm (Figure 4B, d). Microtubule bundles often became

randomly organized, with no reminiscence of the phragmoplast

array (Figure 4B, e).

Among wild-type microspores, 95% demonstrated normal

phragmoplast arrays with mirrored microtubule sets during their

first cytokinesis (n¼ 56). Only 26% of mutant microspores, how-

ever, showed similar phragmoplast arrays at comparable cyto-

kinesis stages as wild-type microspores (n ¼ 118).

Mutant Microspores Failed to Form the Cell Plate

Earlier reports on cytokinesis mutants revealed that fragments of

cell plate–like structures were still formed even though cytoki-

nesis failed (Lauber et al., 1997). In those mutants, cells under-

going cytokinesis have microtubules organized into the mirrored

phragmoplast array. Here, we report that in kinesin-12a kinesin-12b

homozygous double mutants, microtubules no longer bear the

typical phragmoplast array. Thus, we wanted to examine whether

the defective mutant microspores assembled the cell plate

when microtubules were disorganized. Immunolocalization of

KNOLLE, a syntaxin-like protein localized at the cell plate (Lauber

et al., 1997), was performed in both wild-type and mutant

microspores. In the wild-type microspore bearing a mature

phragmoplast, KNOLLE was densely accumulated at the division

site (Figure 5). In the mutant microspore bearing aberrantly

organized microtubules, KNOLLE had a diffuse localization pat-

tern among microtubule bundles (Figure 5). This result suggests

that the accumulation of KNOLLE-bearing vesicles at the division

Figure 3. Alteration of At Kinesin-12 Expression by T-DNA Insertional

Mutations.

(A) Absence of the Kinesin-12A and/or Kinesin-12B transcripts in single

and double homozygous mutants by RT-PCR. The transcripts were

detected in the wild type (12A/12A; 12B/12B), and either transcript was

detected in mutants bearing either one or two copies of the wild-type

Kinesin-12A or Kinesin-12B gene (12a/12a; 12B/12B, 12A/12A; 12b/12b,

12a/12a; 12B/12b, and 12A/12a; 12b/12b). The At1g13320 transcript

encoding protein phosphatase 2A (PP2A) was used as a positive control.

(B) Localization of At Kinesin-12A in dividing microspores of the wild type

(12A 12B) and the double mutant (12a 12b). The At Kinesin-12A signal is

pseudocolored green, and DNA is pseudocolored red. While in the wild

type, microspore-specific signals (white arrowheads) were detected

between the vegetative nucleus (blue arrows) and the generative nucleus

(purple arrows), no such signal was detected in the microspore of the

double mutant. The peripheral signal was due to the autofluorescence of

the pollen coat. Bars ¼ 5 mm.

Phragmoplast Kinesins in Plant Cytokinesis 5 of 11

site is dependent on the antiparallel microtubule array of the

phragmoplast, in which the plus ends of microtubules face each

other in the middle. Hence, in the defective mutant microspores,

disorganized microtubules, when their plus ends were not placed

in the middle of the phragmoplast, prevented KNOLLE from

accumulating at the cell division site.

Callose deposition in developing pollen grains of the double

mutant was also compared with that in wild-type pollen. After

mitosis was completed in wild-type microspores, callose was

deposited between two reforming nuclei (Figure 5B). Eventually,

a complete callose-rich cell plate was formed (Figure 5A). In the

double mutant, defective pollen grains bearing two identical

nuclei lacked callose deposition between the nuclei (Figure 5B).

Instead, callose was detected at the cell cortex as a large aggre-

gate that did not resemble the cell plate (Figure 5B).

DISCUSSION

Our results demonstrate that two highly homologous kinesins,

Kinesin-12A and Kinesin-12B, serve as dynamic integrators of

antiparallel microtubules in the phragmoplast in Arabidopsis.

Simultaneous inactivation of both kinesins by genetic means

often led to loss of the antiparallel pattern of the phragmoplast

microtubule array, which consequently caused the failure of cell

plate formation and cytokinesis in dividing microspores. Thus,

by acting at the plus end of phragmoplast microtubules, the

Kinesin-12A/B motors allow the microtubules without direct

contact to remain in two mirrored sets so that transport from

both sides of the dividing cell takes place in a unidirectional

manner toward the division plane.

The Organization of Phragmoplast Microtubules

Upon the completion of sister chromatid segregation, newly

polymerized microtubules coalesce in the spindle midzone and

later form the phragmoplast microtubule array (Zhang et al.,

1993). Microtubule-associated proteins, known as MAPs, play a

regulatory role in microtubule organization (Lloyd and Hussey,

2001; Jurgens, 2005). A group of evolutionarily conserved MAPs

Figure 4. Comparisons of Microtubule Organization and Cell Plate Development during the First Mitotic Cell Division in the Microspore.

Microtubules are pseudocolored green, and DNA is pseudocolored blue.

(A) In wild-type microspores (a), upon the completion of mitosis, microtubules were organized into an antiparallel array between two DNA masses. A

typical phragmoplast microtubule array (b) had a dark line in the middle. The phragmoplast microtubule array (c) appeared in a barrel-like shape at this

late stage of cytokinesis.

(B) In defective microspores of the homozygous double mutant, microtubules failed to be organized into an antiparallel phragmoplast array.

Microtubules (a) polymerized into bundles between two DNA masses after mitosis. More microtubules were formed between two reforming nuclei (b),

and they did not appear to have a dark line by tubulin immunofluorescence in the middle (c). Aggregates/bundles of microtubules ([d] and [e]) remained

to be associated with one nucleus toward the periphery. Bar ¼ 5 mm.

6 of 11 The Plant Cell

with molecular masses of ;65 kD, known as MAP65/Ase1p/

PRC1, have been shown to decorate phragmoplast microtubules

(Jiang and Sonobe, 1993; Hussey et al., 2002). Particular iso-

forms of MAP65s show discrete patterns of localization in the

phragmoplast (e.g., preferentially toward the plus or the minus

end of phragmoplast microtubules) (Smertenko et al., 2000;

Muller et al., 2004; Van Damme et al., 2004). By exclusively

decorating the spindle midzone at late anaphase and the phrag-

moplast midzone, At MAP65-3/PLE contributes to limiting the

dimension of the phragmoplast microtubule array, as loss-of-

function mutations lead to expansion of the phragmoplast mi-

crotubule midzone (Muller et al., 2004). Separately, tobacco

(Nicotiana tabacum) MAP65-1 was shown to be a substrate of a

phragmoplast-specific mitogen-activated protein kinase cas-

cade, and its phosphorylation is required for the timely depoly-

merization of phragmoplast microtubules (Sasabe et al., 2006).

The other likely organizing factor of phragmoplast microtu-

bules is the MOR1/GEM1 protein in the XMAP215/Dis1/TOG

family (Hussey et al., 2002). The gem1-2 and mor1 mutations at a

locus encoding an evolutionarily conserved XMAP125-like MAP

also leads to a failure of cytokinesis in the microspore and

abnormal cell plate formation in somatic cells (Whittington et al.,

2001; Twell et al., 2002; Eleftheriou et al., 2005; Kawamura et al.,

2006). But the antiparallel pattern of phragmoplast microtubules

is not altered in vegetative cells of the mor1 mutant (Eleftheriou

et al., 2005; Kawamura et al., 2006).

While the aforementioned MAPs likely contribute to the gen-

eral operation of phragmoplast microtubules, their functions may

be limited to regulation of the stability and/or dynamics of the

microtubules. In other words, unlike Kinesin-12A/B, they are

unlikely to contribute to establishing the fundamental pattern of

the phragmoplast microtubule array.

Specialized Roles of Distinct Kinesins in Plant Cytokinesis

Besides Kinesin-12A/B, other kinesins have also been found to

actively participate in cytokinesis in Arabidopsis and other plants

(Lee and Liu, 2004). Those in the Kinesin-5/BIMC subfamily are

among the most well characterized. Kinesin-5 in both tobacco

and carrot (Daucus carota) cells decorates phragmoplast micro-

tubules with an emphasis toward the plus end and plays a role in

microtubule–microtubule sliding (Asada et al., 1997; Barroso

et al., 2000). Functions of similar kinesins have yet to be tested in

Arabidopsis by genetic means.

Conversely, members in the Kinesin-7 subfamily exhibit spa-

tially and temporally specific localization in the middle region of

the phragmoplast. Among them, the NACK1/HIK kinesin inter-

acts physically with a cytokinesis-important mitogen-activated

protein kinase cascade to allow a mitogen-activated protein

kinase kinase kinase to be targeted to the division site and is

required for cytokinesis (Nishihama et al., 2002; Strompen et al.,

2002). A similar kinesin, NACK2/TES, is required exclusively for

male meiotic cytokinesis (Yang et al., 2003). An identified func-

tion of this kinesin–mitogen-activated protein kinase alliance is to

downregulate Nt MAP65-1’s microtubule-bundling activity by

phosphorylation, which is required for the timely execution of

cytokinesis in tobacco cells (Sasabe et al., 2006). But the

Figure 5. The Cell Plate Failed To Be Formed in Defective Microspores.

(A) Localization of the cell plate marker KNOLLE in developing pollen

grains of the wild type and the double mutant. In a wild type (12A 12B)

microspore, the developing cell plate marked by the syntaxin-like protein

KNOLLE (arrowheads) was formed in the middle region of the phragmo-

plast. A dark midline was clearly seen by tubulin immunofluorescence

(arrowheads). In defective microspores of a homozygous double mutant

(12a 12b), microtubules failed to be organized into a phragmoplast array

with a dark midline. KNOLLE accumulated around microtubules in a

diffuse fashion. The peripheral signal was due to the autofluorescence of

the pollen coat. Bars ¼ 10 mm.

(B) Callose accumulation in the wild type and the double mutant. In the

wild type, callose (small arrowheads), labeled by aniline blue, appeared

between the reforming vegetative nucleus (large arrowhead) and the

generative nucleus (arrow) stained by DAPI. The completion of cytoki-

nesis left a callose-rich cell plate (arrowheads) separating the cytoplasms

of the generative cell and the vegetative cell (top right). In defective pollen

grains in the double mutant, callose accumulated as a large aggregate at

the cell cortex (small arrowhead), while two identical nuclei (large arrow-

heads) were positioned away from the aggregate. Such a callose-rich

aggregate (arrowhead) was not organized in a cell plate–like configura-

tion at the cell cortex (bottom right). Bar ¼ 10 mm.

Phragmoplast Kinesins in Plant Cytokinesis 7 of 11

inactivation of the NACK1/HIK kinesin does not alter the pattern

of the phragmoplast microtubule array (Nishihama et al., 2002).

Other kinesins have been implicated in the spatial regulation of

cytokinesis in Arabidopsis. A novel kinesin, as a cyclin-dependent

kinase substrate, localizes to the division site and cortex except

for the site once occupied by the preprophase band (Vanstraelen

et al., 2006). How this intriguing localization pattern might be

linked to phragmoplast operation awaits further examination.

In addition, two novel extra-large kinesins, POK1 and POK2,

play a redundant role in guiding phragmoplast and cell plate orien-

tation during cytokinesis (Muller et al., 2006). However, unlike At

Kinesin-12A/B, these kinesins again do not affect the overall

antiparallel organization of phragmoplast microtubules.

Kinesin-12 as a Microtubule Plus End–Associated Motor

in the Cell Plate Assembly Matrix

As demonstrated by electron microscopic tomography, antipar-

allel phragmoplast microtubules do not interdigitate, and plus

ends of one group of these microtubules are inserted in an

amorphous structure termed the cell plate assembly matrix at the

cell division site in dividing somatic cells (Austin et al., 2005).

Because a dark line was revealed by antitubulin immunofluores-

cence in the phragmoplast of dividing microspores (Figure 4A, b

and c), the microtubules were probably organized as in somatic

cells. It has been postulated that microtubule-stabilizing factors

like EB1 could have contributed to capturing the blunt, metasta-

ble plus ends of these microtubules within the matrix (Austin

et al., 2005). Unfortunately, the composition of the proposed

microtubule plus end–capturing complex has not been deter-

mined.

Our results have provided evidence that by acting directly at

the microtubule plus end, Kinesin-12A/B play a critical role in

allowing phragmoplast microtubules to be organized in two

mirrored sets with a gap between them (Figure 6A). We suggest

that these motors likely are part of the microtubule plus end–

capturing complex in the phragmoplast. However, the mecha-

nism that regulates the temporally specific association of

Kinesin-12 with the plus end of phragmoplast microtubules is

not clear. The kinesin may be targeted there by interacting with

Figure 6. Models of the Function of Kinesin-12.

(A) Kinesin-12A/B and their putative anchoring factor(s) form a protein complex that interacts with the plus ends of phragmoplast microtubules located

in the middle region. They function in translocating newly polymerized microtubule segments and allow the plus ends to be stably located in the middle

region.

(B) The presence of Kinesin-12A/B allows the formation and maintenance of the antiparallel phragmoplast microtubule array. Consequently, successful

cytokinesis brings about the cell plate (red), which separates the generative cell from the vegetative cytoplasm. The generative cell undergoes mitosis to

produce two sperm cells. The absence of Kinesin-12A/B causes microtubules to be bundled together with mixed polarities. Consequently, materials for

cell plate formation do not accumulate in the middle region. Ultimately, two nuclei are suspended in the vegetative cytoplasm. Microtubules are shown

in green, and nuclei are shown in blue.

8 of 11 The Plant Cell

certain anchoring factor(s) residing in the cell plate assembly

matrix. Such a targeting mechanism has been reported for the

XKLP2 kinesin in Xenopus (Wittmann et al., 2000). Together with

such a putative anchoring factor, Kinesin-12A/B can stabilize the

plus ends of the phragmoplast microtubules while still allowing

tubulin dimers to be added to the plus ends. Kinesin-12 would

then drive newly assembled microtubule segments to be trans-

located in an outward manner by acting as a plus end–directed

motor.

During male gametogenesis, the cytoplasm of the generative

cell is physically separated from that of the vegetative cell, as a

result of cell plate formation via the phragmoplast (Figure 6B). To

date, mutations in genes including TIO, encoding a member of

the FUSED kinase family, which acts at the phragmoplast

midzone, lead to the failure of cytokinesis after microspore

mitosis (Oh et al., 2005). However, it has not been observed

that the reported mutations cause a loss of the bipolarity of the

phragmoplast microtubule array. Because mutants such as

those bearing null tio mutations produce a partial cell plate,

one would predict that at least an initial phragmoplast array is

established, which might fail to expand. In the absence of

Kinesin-12A/B, microtubules frequently fail to be organized into

a mirrored phragmoplast array. Consequently, defective micro-

spores failed to produce the generative cell because of the failure

of cell plate formation (Figure 6B).

The fact that some microspores still divide normally in the

double mutant suggests that there are other factor(s) that play a

redundant role like Kinesin-12A/B. In addition, although both

kinesins also decorate the plus end of phragmoplast microtu-

bules in somatic cells (Pan et al., 2004), we did not observe any

defect in cytokinesis during vegetative growth in the homozy-

gous double mutant. Thus, it further suggests that one or more of

the remaining 59 kinesins encoded by the Arabidopsis genome

(Reddy and Day, 2001) may play a redundant role like Kinesin-

12A/B. We suggest that each of these functionally related

kinesins contributes to establishing the phragmoplast microtu-

bule array quantitatively. A qualitative effect is generated when

quantitative functions of individual kinesins are combined. In the

homozygous double mutant reported here, a significant number

of pollen grains were defective despite the fact that no noticeable

defect in diploid cells was detected.

In summary, our results demonstrate that the failure of cyto-

kinesis caused by the inactivation of Kinesin-12 resulted directly

from the disorganization of phragmoplast microtubules. Such a

direct connection between kinesin motors and cell plate forma-

tion establishes the significance of proper microtubule organi-

zation in plant cytokinesis.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana plants bearing T-DNA insertion mutations were

either the Wassilewskija ecotype (kinesin-12a-1 and kinesin-12a-2) or

Columbia (kinesin-12b-1 and kinesin-12b-2). The kinesin-12a-1 and

kinesin-12b-1 lines were reported previously (Pan et al., 2004). The

kinesin-12a-2 line was recovered from the collection at the Arabidop-

sis Knockout Facility of the University of Wisconsin Biotechnology

Center. The kinesin-12b-2 (SALK_027020) line was recovered from the

Sequence-Indexed Library of Insertion Mutations in the Arabidopsis Ge-

nome at the Salk Institute Genome Analysis Laboratory. Seedlings were

grown under 24 h of light at 228C and 70% RH. Standard genetic crosses

were performed between mutant lines. Progeny from crosses between

wild-type Wassilewskija and Columbia plants were used as controls.

PCR-Based Screening

Positive T-DNA insertions were confirmed by a PCR-based method

(Krysan et al., 1996). Primers for kinesin-12a-2 were the gene-specific

primer YT3 (59-TACATGTCAGTAAAAGGGTAATGCAATCA-39) and the

T-DNA border–specific primer JL202 (59-CATTTTATAATAACGCTGCGGA-

CATCTAC-39) for testing T-DNA insertion and the gene-specific primers

3726F2 (59-GATGTTTACCACAAGATGAAATTATCAAC-39) and 3726R

(59-GCTTCTGTAACTAAATTTTCTCCTTCAC-39) for testing homozygosity.

Primers for kinesin-12b-2 were YBTA1 (59-CTATGGGATTTTGTGGCTC-

TGC-39) and the T-DNA border–specific primer LBa1 (59-ATGGTTCA-

CGTAGTGGGCCATC-39) for detecting the T-DNA insertion and the

gene-specific primers YBTA1 and JPF (59-TTAGAAGTTTATTGAATCAA-

TGCAGATATG-39) for testing homozygosity.

RNA Extraction and RT-PCR

Total RNA was isolated from flower buds using PureLink Plant RNA

reagent (Invitrogen) as described by the manufacturer. The expression of

RNA was detected using PCR amplification of reverse transcription

products. Potential genomic DNA contaminants of RNA samples were

eliminated by digesting with DNase I before the reverse transcription

step. The primers used for RT-PCR were PA1-5 (59-GCTGGAGAGT-

TACTTGTTCGG-39) and PA1-3 (59-TCCATTGCTGCTCACTACTTG-39)

for Kinesin-12A and PA1L-5 (59-TGTTCAAGCAGCAGGAGAGTTAC-39)

and PA1L-3 (59-GCCATAGCATCGTCATTACAAGAAG-39) for Kinesin-12B.

RT-PCR of At1g13320, which encodes a subunit of Ser/Thr protein phos-

phatase 2A, served as a positive control (Czechowski et al., 2005). After

40 amplification cycles, PCR products were analyzed by gel electro-

phoresis.

Fluorescence Microscopy

Nuclei in microspores were stained with the dye DAPI according to a

published protocol (Park et al., 1998). Immunolocalization experiments

were performed according to a published study (Terasaka and Niitsu,

1990). Briefly, developing pollen grains were mechanically released from

anthers and fixed with 4% formaldehyde for 1 h at room temperature.

Fixed pollen grains were collected by centrifugation at 3000 rpm for 5 min

and then digested with 1% Cellulase RS and 1% Pectolyase Y-23 (both

from Yakult Honsha) in 50 mM PIPES buffer, pH 5.3, for 2 h with gentle

rocking. The pollen grains were then immobilized on poly-L-Lys–coated

slides prior to incubation with antibodies. Microtubules were labeled by

the DM1A anti-a-tubulin antibody diluted at 1:400 (Sigma-Aldrich); At

Kinesin-12A/B were stained by anti-PAKRP1-C at 1:400 (Lee and Liu,

2000); and KNOLLE was stained with the anti-KNOLLE antibodies at

1:100 (Rose Biotechnology). Wide-field fluorescence images were ac-

quired with a CCD camera (Hamamatsu Photonics) using the Image-

ProPlus 4.0 software package (Media Cybernetics) on an Eclipse E600

microscope equipped with epifluorescence optics (Nikon). Confocal

images were collected with a TCS-SP laser scanning confocal micro-

scope (Leica) using argon and krypton lasers. Images were assembled in

the Adobe Photoshop 7.0 software package.

Developing pollen grains from wild-type and mutant flowers were

stained for callose with aniline blue solution (0.05% [w/v] in 100 mM

potassium phosphate buffer, pH 8.5) for 5 min and observed by fluores-

cence microscopy as described elsewhere (Park and Twell, 2001).

Phragmoplast Kinesins in Plant Cytokinesis 9 of 11

Conventional transmission electron microscopy was performed accord-

ing to a published protocol (Park and Twell, 2001) using the low-viscosity

Spurr mini kit (Ted Pella). Samples were observed with a JEM-100S

transmission electron microscope (JEOL).

Accession Numbers

The Arabidopsis Genome Initiative locus identifiers for the major

genes mentioned in this study are as follows: Kinesin-12A (At4g14150),

Kinesin-12B (At3g23670), and protein phosphatase 2A (At1g13320).

ACKNOWLEDGMENTS

We are grateful to Haihong Liu for her assistance in transmission

electron microscopy and to Sally Assmann for her insightful suggestions

on the manuscript. We thank the ABRC, the University of Wisconsin

Biotechnology Center, the Salk Institute, and the Syngenta Torrey Mesa

Research Institute for providing mutant screening services. Critical

comments made by anonymous reviewers were greatly appreciated.

This work was supported in part by the National Research Initiative of

the USDA Cooperative State Research, Education, and Extension

Service (Grant 2005-35304-16031 to Y.-R.J.L.) and the Energy Biosci-

ences Program of the U.S. Department of Energy (Grant DE-FG02-

04ER15554 to B.L.).

Received January 26, 2007; revised July 29, 2007; accepted August 6,

2007; published August 24, 2007.

REFERENCES

Asada, T., Kuriyama, R., and Shibaoka, H. (1997). TKRP125, a kinesin-

related protein involved in the centrosome-independent organization

of the cytokinetic apparatus in tobacco BY-2 cells. J. Cell Sci. 110:

179–189.

Asada, T., Sonobe, S., and Shibaoka, H. (1991). Microtubule translo-

cation in the cytokinetic apparatus of cultured tobacco cells. Nature

350: 238–241.

Austin, J.R., Segui-Simarro, J.M., and Staehelin, L.A. (2005). Quan-

titative analysis of changes in spatial distribution and plus-end ge-

ometry of microtubules involved in plant-cell cytokinesis. J. Cell Sci.

118: 3895–3903.

Barroso, C., Chan, J., Allan, V., Doonan, J., Hussey, P., and Lloyd, C.

(2000). Two kinesin-related proteins associated with the cold-stable

cytoskeleton of carrot cells: Characterization of a novel kinesin,

DcKRP120–2. Plant J. 24: 859–868.

Boleti, H., Karsenti, E., and Vernos, I. (1996). Xklp2, a novel Xenopus

centrosomal kinesin-like protein required for centrosome separation

during mitosis. Cell 84: 49–59.

Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., and Scheible,

W.R. (2005). Genome-wide identification and testing of superior

reference genes for transcript normalization in Arabidopsis. Plant

Physiol. 139: 5–17.

Eleftheriou, E.P., Baskin, T.I., and Hepler, P.K. (2005). Aberrant cell

plate formation in the Arabidopsis thaliana microtubule organization

1 mutant. Plant Cell Physiol. 46: 671–675.

Hussey, P.J., Hawkins, T.J., Igarashi, H., Kaloriti, D., and Smertenko,

A. (2002). The plant cytoskeleton: Recent advances in the study of the

plant microtubule-associated proteins MAP-65, MAP-190 and the

Xenopus MAP215-like protein, MOR1. Plant Mol. Biol. 50: 915–924.

Jiang, C.J., and Sonobe, S. (1993). Identification and preliminary

characterization of a 65-Kda higher-plant microtubule-associated

protein. J. Cell Sci. 105: 891–901.

Jurgens, G. (2005). Plant cytokinesis: Fission by fusion. Trends Cell

Biol. 15: 277–283.

Kawamura, E., Himmelspach, R., Rashbrooke, M.C., Whittington,

A.T., Gale, K.R., Collings, D.A., and Wasteneys, G.O. (2006).

MICROTUBULE ORGANIZATION 1 regulates structure and function

of microtubule arrays during mitosis and cytokinesis in the Arabidop-

sis root. Plant Physiol. 140: 102–114.

Krysan, P.H., Young, J.C., Tax, F., and Sussman, M.R. (1996).

Identification of transferred DNA insertions within Arabidopsis genes

involved in signal transduction and ion transport. Proc. Natl. Acad.

Sci. USA 93: 8145–8150.

Lauber, M.H., Waizenegger, I., Steinmann, T., Schwarz, H., Mayer,

U., Hwang, I., Lukowitz, W., and Jurgens, G. (1997). The Arabidop-

sis KNOLLE protein is a cytokinesis-specific syntaxin. J. Cell Biol.

139: 1485–1493.

Lee, Y.R.J., and Liu, B. (2000). Identification of a phragmoplast-

associated kinesin-related protein in higher plants. Curr. Biol. 10:

797–800.

Lee, Y.R.J., and Liu, B. (2004). Cytoskeletal motors in Arabidopsis.

Sixty-one kinesins and seventeen myosins. Plant Physiol. 136: 3877–

3883.

Liu, B., and Lee, Y.R.J. (2001). Kinesin-related proteins in plant cyto-

kinesis. J. Plant Growth Regul. 20: 141–150.

Lloyd, C., and Hussey, P. (2001). Microtubule-associated proteins in

plants—Why we need a MAP. Nat. Rev. Mol. Cell Biol. 2: 40–47.

McCormick, S. (2004). Control of male gametophyte development.

Plant Cell 16 (suppl.): S142–S153.

Miki, H., Okada, Y., and Hirokawa, N. (2005). Analysis of the kinesin

superfamily: Insights into structure and function. Trends Cell Biol. 15:

467–476.

Muller, S., Han, S., and Smith, L.G. (2006). Two kinesins are involved in

the spatial control of cytokinesis in Arabidopsis thaliana. Curr. Biol. 16:

888–894.

Muller, S., Smertenko, A., Wagner, V., Heinrich, M., Hussey, P., and

Hauser, M. (2004). The plant microtubule-associated protein

AtMAP65-3/PLE is essential for cytokinetic phragmoplast function.

Curr. Biol. 14: 412–417.

Nishihama, R., Soyano, T., Ishikawa, M., Araki, S., Tanaka, H.,

Asada, T., Irie, K., Ito, M., Terada, M., Banno, H., Yamazaki, Y.,

and Machida, Y. (2002). Expansion of the cell plate in plant cytoki-

nesis requires a kinesin-like protein/MAPKKK complex. Cell 109:

87–99.

Oh, S.A., Johnson, A., Smertenko, A., Rahman, D., Park, S.K.,

Hussey, P.J., and Twell, D. (2005). A divergent cellular role for the

FUSED kinase family in the plant-specific cytokinetic phragmoplast.

Curr. Biol. 15: 2107–2111.

Pan, R., Lee, Y.R.J., and Liu, B. (2004). Localization of two homologous

Arabidopsis kinesin-related proteins in the phragmoplast. Planta 220:

156–164.

Park, S.K., Howden, R., and Twell, D. (1998). The Arabidopsis thaliana

gametophytic mutation gemini pollen1 disrupts microspore polarity,

division asymmetry and pollen cell fate. Development 125: 3789–

3799.

Park, S.K., and Twell, D. (2001). Novel patterns of ectopic cell plate

growth and lipid body distribution in the Arabidopsis gemini pollen1

mutant. Plant Physiol. 126: 899–909.

Reddy, A.S.N., and Day, I.S. (2001). Kinesins in the Arabidop-

sis genome: A comparative analysis among eukaryotes. BMC Geno-

mics 2: 2.

Sasabe, M., Soyano, T., Takahashi, Y., Sonobe, S., Igarashi, H., Itoh,

T.J., Hidaka, M., and Machida, Y. (2006). Phosphorylation of

10 of 11 The Plant Cell

NtMAP65-1 by a MAP kinase down-regulates its activity of microtu-

bule bundling and stimulates progression of cytokinesis of tobacco

cells. Genes Dev. 20: 1004–1014.

Smertenko, A., Saleh, N., Igarashi, H., Mori, H., Hauser-Hahn, I.,

Jiang, C.-J., Sonobe, S., Lloyd, C.W., and Hussey, P.J. (2000). A

new class of microtubule-associated proteins in plants. Nat. Cell Biol.

2: 750–753.

Staehelin, L.A., and Hepler, P.K. (1996). Cytokinesis in higher plants.

Cell 84: 821–824.

Strompen, G., El Kasmi, F., Richter, S., Lukowitz, W., Assaad, F.F.,

Jurgens, G., and Mayer, U. (2002). The Arabidopsis HINKEL gene

encodes a kinesin-related protein involved in cytokinesis and is

expressed in a cell cycle-dependent manner. Curr. Biol. 12: 153–158.

Terasaka, O., and Niitsu, T. (1990). Unequal cell division and chromatin

differentiation in pollen grain cells. II. Microtubule dynamics associ-

ated with the unequal cell division. Bot. Mag. (Tokyo) 103: 133–142.

Twell, D., Park, S.K., Hawkins, T.J., Schubert, D., Schmidt, R.,

Smertenko, A., and Hussey, P.J. (2002). MOR1/GEM1 has an

essential role in the plant-specific cytokinetic phragmoplast. Nat.

Cell Biol. 4: 711–714.

Van Damme, D., Bouget, F., Van Poucke, K., Inze, D., and Geelen, D.

(2004). Molecular dissection of plant cytokinesis and phragmoplast

structure: A survey of GFP-tagged proteins. Plant J. 40: 386–398.

Vanstraelen, M., Van Damme, D., De Rycke, R., Mylle, E., Inze, D.,

and Geelen, D. (2006). Cell cycle-dependent targeting of a kinesin at

the plasma membrane demarcates the division site in plant cells. Curr.

Biol. 16: 308–314.

Vantard, M., Levilliers, N., Hill, A.M., Adoutte, A., and Lambert, A.M.

(1990). Incorporation of Paramecium axonemal tubulin into higher

plant cells reveals functional sites of microtubule assembly. Proc.

Natl. Acad. Sci. USA 87: 8825–8829.

Whittington, A.T., Vugrek, O., Wei, K.J., Hasenbein, N.G., Sugimoto,

K., Rashbrooke, M.C., and Wasteneys, G.O. (2001). MOR1 is

essential for organizing cortical microtubules in plants. Nature 411:

610–613.

Wittmann, T., Wilm, M., Karsenti, E., and Vernos, I. (2000). TPX2, a

novel Xenopus MAP involved in spindle pole organization. J. Cell Biol.

149: 1405–1418.

Yang, C., Spielman, M., Coles, J., Li, Y., Ghelani, S., Bourdon, V.,

Brown, R., Lemmon, B., Scott, R., and Dickinson, H. (2003).

TETRASPORE encodes a kinesin required for male meiotic cytokine-

sis in Arabidopsis. Plant J. 34: 229–240.

Zhang, D., Wadsworth, P., and Hepler, P.K. (1993). Dynamics of

microfilaments are similar, but distinct from microtubules during

cytokinesis in living, dividing plant cells. Cell Motil. Cytoskeleton 24:

151–155.

Zhang, D.H., Wadsworth, P., and Hepler, P.K. (1990). Microtubule

dynamics in living dividing plant cells—Confocal imaging of micro-

injected fluorescent brain tubulin. Proc. Natl. Acad. Sci. USA 87:

8820–8824.

Phragmoplast Kinesins in Plant Cytokinesis 11 of 11

DOI 10.1105/tpc.107.050716; originally published online August 24, 2007;Plant Cell

Yuh-Ru Julie Lee, Yan Li and Bo LiuMale Gametogenesis

Phragmoplast-Associated Kinesins Play a Critical Role in Cytokinesis duringArabidopsisTwo

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