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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.
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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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