Arabidopsis Kinesin KP1 Specifically Interacts with VDAC3, aMitochondrial Protein, and Regulates Respiration during SeedGermination at Low Temperature W OA

Xue-Yong Yang,1 Zi-Wei Chen,1 Tao Xu, Zhe Qu, Xiao-Di Pan, Xing-Hua Qin, Dong-Tao Ren,2 and Guo-Qin Liu2,3

State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing

100193, China

The involvement of cytoskeleton-related proteins in regulating mitochondrial respiration has been revealed in mammalian

cells. However, it is unclear if there is a relationship between the microtubule-based motor protein kinesin and mitochon-

drial respiration. In this research, we demonstrate that a plant-specific kinesin, Kinesin-like protein 1 (KP1; At KIN14 h), is

involved in respiratory regulation during seed germination at a low temperature. Using in vitro biochemical methods and in

vivo transgenic cell observations, we demonstrate that KP1 is able to localize to mitochondria via its tail domain (C

terminus) and specifically interacts with a mitochondrial outer membrane protein, voltage-dependent anion channel 3

(VDAC3). Targeting of the KP1-tail to mitochondria is dependent on the presence of VDAC3. When grown at 48C, KP1dominant-negative mutants (TAILOEs) and vdac3 mutants exhibited a higher seed germination frequency. All germinating

seeds of the kp1 and vdac3 mutants had increased oxygen consumption; the respiration balance between the cytochrome

pathway and the alternative oxidase pathway was disrupted, and the ATP level was reduced. We conclude that the plant-

specific kinesin, KP1, specifically interacts with VDAC3 on the mitochondrial outer membrane and that both KP1 and VDAC3

regulate aerobic respiration during seed germination at low temperature.

INTRODUCTION

Much of the aerobic oxidation in eukaryotic cells takes place in

mitochondria. A number of studies have shown that microfila-

ments and microtubules function in mitochondrial movement

and positioning in eukaryotic cells (Hirokawa, 1998). Cytoskel-

etal proteins are also involved in regulating the permeability

of the mitochondrial outer membrane to ADP in animal cells

(Rappaport et al., 1998; Saks et al., 1995). It is well known that

the membrane permeability of mitochondria is mainly depen-

dent on the voltage-dependent anion channel (VDAC) (also

named as a porin), the most abundant integral membrane

protein in the mitochondrial outer membrane (Benz, 1994;

Colombini, 1979; Liu and Colombini, 1992). Recently, both

tubulin and actin from human and yeast (Saccharomyces

cerevisiae) cells were found to interact with VDACs (Carre

et al., 2002; Roman et al., 2006). In vitro reconstitution studies

demonstrated that fungal VDACs have two main conductance

states: an open state that allows the diffusion of large metab-

olites, including nucleotides, and a closed state that regulates

ATP flux through the membrane (Rostovtseva and Colombini,

1996). Based on patch-clamp and planar lipid bilayer tech-

niques, Rostovtseva et al. (2008) demonstrated that tubulin

could induce reversible blockage of VDACs and decrease ATP/

ADP permeability through the mitochondrial outer membrane

and that the addition of tubulin in the reaction system could

reduce the respiration rate of mitochondria (Rostovtseva et al.,

2008). Genomic sequence analysis revealed that there are

five VDAC isoforms in Arabidopsis thaliana, four of which have

been cloned and identified (Clausen et al., 2004). However, it is

still not known whether plant VDACs interact with cytoskeletal

proteins.

We identified a plant-specific kinesin member in Arabidopsis,

Kinesin-like protein 1 (KP1) (standardized nomenclature: At

KIN14h; see Malcos and Cyr, 2009), and found that it binds to

mitochondria based on immunoblot analysis (Ni et al., 2005). The

motor domain of KP1 has nucleotide-dependent microtubule

binding ability and microtubule-stimulated ATPase activity (Li

et al., 2007). Kinesins constitute a superfamily of microtubule

motor proteins and play critical roles in the transport of vesicles

and organelles, cytokinesis, morphogenesis, and signal trans-

duction (Reddy, 2001; Verhey et al., 2001; Lee and Liu, 2004;

Hirokawa et al., 2009). Several animal kinesins, such as KIF1B

and KIF5B in mouse cells (Nangaku et al., 1994; Tanaka et al.,

1998) and KLP67A in early Drosophila melanogaster embryos

(Pereira et al., 1997), have been implicated in the movement of

mitochondria. Green fluorescent protein (GFP) fusion and tran-

sient expression assays showed that two Arabidopsis kinesins,

MKRP1 and MKRP2, were expressed in mitochondria via their

N-terminal mitochondrial targeting signals (Itoh et al., 2001). It is

1 These authors contributed equally to this work.2Guo-Qin Liu and Dong-Tao Ren’s groups contributed equally to thiswork.3 Address correspondence to liu@cau.edu.cn.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Guo-Qin Liu(liu@cau.edu.cn).WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.110.082420

The Plant Cell, Vol. 23: 1093–1106, March 2011, www.plantcell.org ã 2011 American Society of Plant Biologists

not currently understood if kinesins are involved in regulating

mitochondrial functions in plant cells.

The membrane-associated electron transport chain of plant

mitochondria has unique features, such as the ubiquitous pres-

ence of a terminal alternative oxidase (AOX), an important

member of the cyanide (CN)-resistant pathway that competes

for electrons with the standard cytochrome pathway (Laties,

1982; Finnegan et al., 2004) and is able to reduce the levels of

reactive oxygen species (Maxwell et al., 1999; Umbach et al.,

2005). Therefore, the respiratory regulation of plant mitochondria

is expected to have unique characteristics. What is the interac-

tion protein of KP1 in the mitochondria? Does the microtubule-

based motor protein, KP1, function in mitochondrial respiration?

It is of crucial importance to elucidate these questions to reveal

the regulation mechanisms of plant mitochondrial respiration. In

this study, we found that KP1 specifically interacts with the

mitochondrial outer membrane protein VDAC3 and that both

KP1 and VDAC3 are involved in keeping the ATP levels stable

and balancing the aerobic respiration pathways during seed

germination at low temperature (48C).

RESULTS

The Tail Domain Is Responsible forMitochondrial Targeting

of KP1

According to our previous work, KP1 is found in isolated mito-

chondria (Ni et al., 2005). Based on the sequence alignment, we

know that the tail domain of KP1 (KP1-tail; 749 to 1087 amino

acids) is specific among all Arabidopsis kinesins. Tail domains

of many animal kinesins are responsible for cargo binding

(Hirokawa et al., 2009). To gain insight into the molecular mech-

anism underlying the interaction between KP1 and mitochon-

dria, GFP-KP1 and its truncated proteins, Dtail (1 to 748 amino

acids) with GFP at its N terminus, and tail (749 to 1087 amino

acids) with GFP at its C terminus (Figure 1A) were transiently

expressed in Arabidopsis protoplasts prepared from suspension

cells. By immunolabeling microtubules and microfilaments in

the transfected protoplasts and treating them with microtubule/

microfilament-depolymerizing drugs, oryzalin and latrunculin B,

respectively, we found that in addition to localizing to dot-like

Figure 1. The Tail Domain Is Responsible for Mitochondrial Targeting of KP1 in Arabidopsis Protoplasts.

(A) GFP fusion constructs of KP1 and its truncated variants (GFP-Dtail and tail-GFP). The domains of KP1 are indicated by head (head domain; 1 to 374

amino acids), motor (motor domain; 375 to 748 amino acids), and tail (tail domain; 749 to 1087 amino acids).

(B) Immunofluorescence labeling of microtubules and microfilaments in Arabidopsis suspension cell protoplasts transiently overexpressing GFP-KP1.

For microtubule labeling, anti-a-tubulin antibody and tetramethylrhodamine b-isothiocyanate–conjugated goat anti-mouse IgG were used. For

microfilaments, rhodamine-phalloidin was used. Bars = 5 mm.

(C) KP1 fusion proteins in suspension cell protoplasts are able to localize to mitochondria via their tail domain. From left to right, green channel

(GFP), red channel (MitoTracker, mitochondria selective reagent), merged images of green and red channel (Merge), and transmission images

(Bright). Bars = 5 mm.

1094 The Plant Cell

organelles, GFP-KP1 localized to microtubules (Figure 1B; see

Supplemental Figure 1 online). Costaining the protoplasts with

the mitochondrion-selective reagent MitoTracker Red revealed

that some dot-like signals of GFP-KP1 colocalized with mito-

chondria (Figure 1C, white arrows). Interestingly, tail-GFP was

located in mitochondria, but GFP-Dtail was distributed randomly

(Figure 1C). This indicates that KP1 is able to target to the

mitochondria via its tail domain.

To confirm the mitochondrial targeting of the tail domain

of KP1, the constructs for the stable expression of tail-GFP

were fused to the cauliflower mosaic virus 35S promoter (Fig-

ure 2A) and transformed into Arabidopsis. Two overexpression

lines (TAILOE1 and TAILOE2) were identified by immunoblot-

ting with a monoclonal antibody against GFP (Figure 2B). The

results indicate that tail-GFP indeed localizes to mitochondria

(Figure 2C).

KP1 Specifically Interacts with VDAC3

To identify if the tail of KP1 interacts with mitochondrial proteins,

we performed a yeast two-hybrid screen using the tail polypep-

tide as bait. A few polypeptides were identified as potential

interaction partners, of which we focused on a mitochondrial

outer membrane protein, VDAC3. The interaction between KP1

and VDAC3 was then further analyzed using the different do-

mains of KP1 in the yeast two-hybrid system (Figure 3A). Both

KP1-tail and KP1-tail200 (888 to 1087 amino acids) could interact

with VDAC3 to induce Trp1, Leu2,His3, Ade2, and LacZ reporter

genes in AH109 yeast lines, but the N-terminal domain (KP1-N;

1 to 373 amino acids) and the motor domain (KP1-M; 374 to 749

amino acids) of KP1 could not (Figure 3A). To determine if KP1

interacts with other members of the VDAC family in Arabidopsis,

VDAC1, VDAC2, and VDAC4 were cloned and transformed into

yeast cells. The results show that only VDAC3 interacts with KP1-

tail (Figure 3B).

The specific interaction between KP1-tail200 and VDAC3 in

vitrowas verified by aGSTpull down (Figure 3C) and a far-protein

gel blot (Figures 3D and 3E). As shown in Figure 3C, the 30-kD

KP1-tail200 associated with the bacterially expressed 60-kD

GST-VDAC3, while the control GST protein did not. For far-

protein gel blot analysis, two kinds of recombinant proteins, His-

VDAC3 (Figure 3D) and GST-VDAC3 (Figure 3E), were bacterially

expressed, isolated by SDS-PAGE, and transferred onto poly-

vinylidene difluoride (PVDF) membranes. After the membranes

were incubatedwith His-KP1-tail200, the anti-KP1 antibodies that

specifically label His-KP1-tail200 (Figure 3D, Immunoblot, lane 4)

not only recognized the His-KP1-tail200 (Figure 3D, far-western,

lane 4) but also the His-VDAC3 protein bands (Figure 3D, far-

western, lanes 2 and 3). Similarly, anti-His antibodies not only

specifically recognized His-KP1-tail200 (Figure 3E, Immunoblot,

lane 39; far-western, lane 39) but also the 60-kD protein band

of GST-VDAC3 (Figure 3E, far-western, lane 2’). Collectively,

we conclude that VDAC3 directly interacts with the KP1- tail200in vitro.

The specific interaction in vivo between KP1 and VDAC3 was

verified by two methods. Bimolecular fluorescence complemen-

tation (BiFC) showed that both KP1 and KP1-tail interact with

VDAC3 (Figure 4A). The interaction occurs specifically at mito-

chondria (Figure 4B), consistent with the localization of GFP-

VDAC3 (Figure 4C). To support these data, the firefly luciferase

complementation imaging (LCI) system was also performed.

When the constructed pairs of KP1-tail-NLuc/CLuc-VDAC3 and

KP1-NLuc/CLuc-VDAC3 were transformed into tobacco (Nico-

tiana tabacum) leaf cells, fluorescence signals were detected

(Figure 4D). All of these results demonstrate that KP1 specifically

interacts with VDAC3.

Figure 2. Mitochondrial Localization and Immunoblotting Test of KP1-Tail in Stem.

(A) The diagram represents the transgenic construct used to overexpress KP1-tail-GFP. GFP was fused to the tail domain of KP1.

(B) Immunoblot identification of tail-GFP in Col, TAILOE1, and TAILOE2 using anti-GFP antibody (1:10,000). Molecular marker is indicated in kilodaltons

in the left margin.

(C) KP1-tail-GFP in the stem of transgenic seedlings colocalized with mitochondria. From left to right, green channel (GFP), red channel (MitoTracker,

mitochondria selective reagent), merged images of green and red channel (Merge), and transmission images (Bright). Bars = 20 mm.

Kinesin and VDAC in Seed Respiration 1095

Mitochondrial Targeting of the KP1 Tail Is Dependent on Its

Interaction with VDAC3

To further analyze the localization of the KP1-tail via VDAC3

and to elucidate the function of KP1 and VDAC3, we isolated the

T-DNA insertion mutants of KP1 (kp1-1 and kp1-2) (Figure 5A)

and VDAC3 (vdac3-1 and vdac3-2) (Figure 5B) in the Columbia

(Col) background. In two kp1mutants, the T-DNA inserted within

the exon; in vdac3-1, the T-DNA inserted in the 59-untranslated

region; and in vdac3-2, the T-DNA inserted in the fourth intron

of the gene. The homozygous knockout mutants were isolated

and confirmed by PCR analysis, and their gene expression

was analyzed using RT-PCR. As shown in Figure 5C, the ex-

pression of KP1 was completely interrupted by the T-DNA

insertion in the kp1 mutants, and VDAC3 expression was signif-

icantly reduced in vdac3 mutants (Figure 5D).

We then tested the localization of tail-GFP in vdac3-1mutants

and GFP-VDAC3 in kp1-1 mutants to establish whether the

interaction between KP1 and VDAC3 confers the mitochondrial

targeting ability of the KP1 tail. As shown in Figure 6, the KP1

knockout did not inhibit the mitochondrial localization of GFP-

VDAC3; however, in vdac3-1 mutants, very little tail-GFP

Figure 3. Identification and Verification of a KP1-Interacting Protein, VDAC3.

(A) Yeast two-hybrid assay to analyze the interaction between KP1, KP1-N (1 to 373 amino acids), KP1-M (374 to 748 amino acids), KP1-tail (749 to

1087 amino acids), and KP1-tail200 (888 to 1087 amino acids) and VDAC3 on 4D agar medium (-Trp, -Leu, -His, -Ade). Only KP1-tail and KP1-tail200show binding activity with VDAC3.

(B) Yeast two-hybrid assay to analyze the interaction between four VDAC isoforms from Arabidopsis and KP1-tail and KP1-tail200. KP1-tail and KP1-

tail200 specifically interact with VDAC3.

(C) GST pull-down assay. GST (27 kD) or GST-VDAC3 (60 kD) protein was bound to beads and then incubated with 63His-tagged KP1-tail200 proteins

(30 kD). The eluted proteins were separated by SDS-PAGE. Anti-KP1 (1:5000) and anti-GST (1:100,000) antibodies were used for immunoblotting.

(D) Far-protein gel blot to verify the interaction between His-VDAC3 and His-KP1-tail200. Lane 1, unrelated His-tagged recombinant protein as negative

control; lane 2, purified His-VDAC3 (30 kD); lane 3, total extracts from Escherichia coli expressing His-VDAC3; lane 4, purified His-KP1-tail200 (30 kD).

The SDS-PAGE gel was stained with Coomassie blue and transformed onto PVDF membranes. The far-protein gel blot was performed with anti-KP1

antibody (1:5000) after the membrane was incubated with His-KP1-tail200. Two immunoblots were performed as positive controls with anti-KP1 (1:5000)

and anti-VDAC3 (1:5000) antibodies, respectively, to confirm the recombinant proteins. Molecular markers are indicated in kilodaltons in the left margin.

(E) Far-protein gel blot for the interaction assay between GST-VDAC3 and His-KP1-tail200. Lanes 1’ and 2’, the total extracts from E. coli expressing

GST and GST-VDAC3, respectively; lane 39, purified His-KP1-tail200. The far-protein gel blot was performed with an anti-His tag monoclonal antibody

after the membrane was incubated with His-KP1-tail200. Two immunoblots were performed as positive controls with anti-His (1:10,000) and anti-VDAC3

(1:5000) antibodies, respectively, to confirm the recombinant proteins. Molecular markers are indicated in kilodaltons in the left margin.

1096 The Plant Cell

colocalized with MitoTracker Red, suggesting that the mito-

chondrial localization of KP1 is dependent on its interaction

with VDAC3.

Both KP1 and VDAC3 Function during Seed Germination

at 48C

The phenotypes of all kp1 mutants and transgenic plants pre-

pared in this study were examined under normal growth condi-

tions throughout the plant’s life, but no visible phenotypes were

found, including regarding the distribution and morphology of

mitochondria (see Supplemental Figure 2 online). According to a

previous bioinformatics approach and RT-PCR analysis, KP1

expression is significantly reduced after cold treatment (Li et al.,

2008) and increased during seed germination (Schmid et al.,

2005). We designed real-time PCR experiments to understand

the gene transcription changes in detail. When wild-type seeds

were germinated at either 22 or 48C, the mRNA level of KP1

exhibited a sudden increase at day 2 or day 4 (Figures 7A and

7B), indicating the involvement of KP1 during seed germination.

Therefore, we decided to investigate the seed germination

phenotypes of the mutant lines resulting from the chilling

Figure 4. KP1 Interacts with VDAC3 in Vivo.

(A) BiFC assay. In Arabidopsis mesophyll protoplasts transformed with the construct pairs KP1-YFPN plus YFPC-VDAC3 or KP1-tail-YFPN plus YFPC-

VDAC3, a YFP fluorescence signal was detected. For protoplasts transformed with the construct pairs KP1-tail-YFPN plus YFPC or YFPN plus YFPC-

VDAC3, no fluorescence signal was detected. Bars = 5 mm.

(B) Colocalization of the KP1 and VDAC3 complex with mitochondria. In Arabidopsis mesophyll protoplasts transformed with the construct pairs KP1-

tail-YFPN and YFPC-VDAC3, YFP fluorescence colocalized with mitochondria stained by MitoTracker. Bars = 5 mm.

(C) Colocalization of VDAC3 with mitochondria. Arabidopsis mesophyll protoplasts transiently overexpressing GFP-VDAC3 were stained with

MitoTracker. Bars = 5 mm.

(D) LCI assay. The tobacco leaves were transformed by infiltration using a needleless syringe with the construct pairs KP1-NLuc/CLuc-VDAC3, KP1-

tail-NLuc/CLuc-VDAC3, KP1-NLuc/CLuc, KP1-tail-NLuc/CLuc, NLuc/CLuc-VDAC3, VDAC3-Nluc/CLuc-KP1, VDAC3-Nluc/CLuc-KP1-tail, and NLuc/

CLuc. Signal fluorescence was detected on the locations infiltrated by the construct pairs KP1-NLuc/CLuc-VDAC3 and KP1-tail-NLuc/CLuc-VDAC3.

The left panel shows the bright-field image of the treated leaf.

Kinesin and VDAC in Seed Respiration 1097

treatment. Interestingly, vdac3 T-DNA insertion lines exhibited

a higher seed germination frequency than the wild type at 48C,especially at day 10 (Figure 8A). After 14 d, all tested seeds

germinated completely, but the seedlings of the mutants were

stronger (Figure 8B) and the roots longer (Figure 8C). However,

no significant difference between kp1 mutants and the wild

type (Col) was observed at 48C, which may be due to gene

redundancy or the subtle function of KP1 during this process.

For this reason, transgenic lines overexpressing the KP1-tail,

TAILOE1 and TAILOE2, were generated and characterized as

dominant-negative mutants (Figures 2A and 2B). We inferred

that in dominant-negative mutants, the overexpressed KP1-

tail could compete with functional KP1 and its functionally redun-

dant proteins in plant cells. As expected, the transgenic lines

showed similar phenotypes to the vdac3 mutants at 48C (Figures

8A to 8C).

To determine if the visual phenotype of the mutants at

48C was due to quicker germination but not faster growth,

seeds were germinated at 228C for 2 d before being transferred

to 48C. Themutants did not present any obviousmorphological

phenotype. These results demonstrate that both KP1 and

VDAC3 function during seed germination at a low temperature.

KP1 and VDAC3 Regulate Respiration Pathways and

ATP Levels

The germination of seeds at the dormant stage requires efficient

energy from degradation metabolism. In a dry seed, the

Figure 5. Isolation of kp1 and vdac3 T-DNA Insertion Lines.

(A) Diagram of the KP1 gene structure. Introns are shown as lines, and exons are shown as black boxes. Triangles indicate the locations of the T-DNA

insertion sites for kp1-1 and kp1-2.

(B) The gene structure of VDAC3 and the T-DNA insertion sites of the vdac3 mutants. Introns are shown as lines, and exons are shown as black boxes.

Triangles indicate the locations of the T-DNA insertion sites.

(C) RT-PCR analysis of the wild type (Col) and kp1mutants. KP1 transcripts are absent in kp1mutants. TheUBQ5 transcript was amplified as an internal

control.

(D) RT-PCR analysis of the wild type (Col) and vdac3 mutants. VDAC3 transcripts are downregulated in vdac3 mutants. The UBQ5 gene was amplified

as an internal control. Similar results were obtained with at least three biological replicates of the experiments.

Figure 6. The KP1-Tail Localized to Mitochondria in a VDAC3-Dependent Manner.

GFP-VDAC3 merged with MitoTracker in Arabidopsismesophyll protoplasts from kp1-1, but the KP1-tail-GFP fusion expressed in vdac3-1 protoplasts

showed little colocalization with mitochondria.

1098 The Plant Cell

mitochondria barely function, and one of the initial changes

during the early stages of germination is the resumption of

respiratory activity (Bewley, 1997). For this reason, the respira-

tory rate of kp1 and vdac3mutantswas investigated. As shown in

Figure 9, oxygen consumption increased significantly in kp1 and

vdac3 mutants during the period of seed germination at 48C,especially in the plants germinated for 7 to 11 d. In higher plants,

the oxygen consumption usually results from the cytochrome

pathway and the AOX pathway in mitochondria; therefore, we

measured the oxygen consumption of seeds germinated at day

10 at 48C in the presence of the specific respiration inhibitors,

NaN3 (for inhibiting the cytochrome pathway) and salicylhy-

droxamic acid (SHAM; for inhibiting the AOX pathway). The

results showed that in kp1 mutants SHAM-resistant respiration

(similar to the cytochrome pathway) increased greatly, while CN-

resistant respiration (similar to the AOX pathway) decreased. The

ratio of oxygen consumption via the cytochrome pathway and

the AOX pathway changed from ;1:1 in the wild type to 6:1 in

kp1 mutants (Figure 10A). The vdac3-1 mutants showed similar

results, with a ratio of ;3:1. We obtained the VDAC3 over-

expression transgenic line (VOE) to confirm the above results

(Figures 10B and 10C). The VOE line restored the oxygen

consumption ratio of the cytochrome pathway to AOX pathway

(Figure 10A).

It is well known that the energy from the mitochondrial cyto-

chrome pathway is mainly used for ATP synthesis. Therefore, we

measured the ATP levels in 10-d-old seedlings. Interestingly, the

kp1 mutants grown at 48C produced ;0.6 nmol ATP/g fresh

weight, which is 31% lower than that in the wild type; the ATP

level in the vdac3-1 mutants also decreased, by 45% (Figure

11A). The restoration of the ATP level in transgenic Arabidopsis

plants overexpressing VDAC3 further confirmed this vdac3 mu-

tant phenotype. To eliminate the possibility that the observed

changes in ATP levels were due to a different quantity of mito-

chondria, the activity of citrate synthase, a marker for mitochon-

drial mass (Moraes et al., 1993), was assayed. No significant

difference between the wild type and the mutants was observed

(Figure 11B).

The SHAM/CN-resistant respiration and ATP levels were

also assayed in the dominant-negative mutants TAILOE1 and

TAILOE2. The ratio of the SHAM-resistant respiration to CN-

resistant respiration was found to be;6:1 (Figure 12A), and the

ATP level was 35% lower than that of Col (Figure 12B). This

is similar to our findings for kp1 mutants, indicating that the

overexpressed tail domain suppressed the function of KP1.

These results demonstrate that both KP1 and VDAC3 are in-

volved in regulating respiration pathways and ATP levels during

seed germination at a low temperature.

DISCUSSION

Kinesins constitute a superfamily of microtubule motor proteins

and are known to be essential for many cellular functions (Miki

et al., 2005). Studies on the kinesins of higher plants revealed

their participation inmicrotubule organization duringmeiosis and

mitosis (Chen et al., 2002; Marcus et al., 2003), cytokinesis (Lee

et al., 2007), cellulose microfibril deposition (Zhong et al., 2002),

and morphogenesis (Oppenheimer et al., 1997; Reddy et al.,

2004; Lu et al., 2005). In this study, a plant-specific kinesin KP1

(At KIN14h) was found to interact with the mitochondrial outer

membrane protein VDAC3 via its tail domain and to be involved in

the regulation of respiration during seed germination at low

temperature.

KP1 Specifically Interacts with the Mitochondrial Outer

Membrane Protein VDAC3

Systematic analysis of the sequences in theArabidopsis genome

with the conservative motor domain of kinesins revealed the

presence of 61 kinesins or kinesin-like proteins (Reddy and Day,

2001). KP1, a member of the subfamily kinesin-14, tightly binds

to mitochondria from Arabidopsis (Ni et al., 2005). Here, using

several biochemical and transgenic methods, we demonstrate

the specific interaction of KP1 with VDAC3, an isoform of

Arabidopsis VDACs that normally localizes to the outer mem-

brane of mitochondria (Colombini, 1979). With the exception of

VDAC5, whose gene has not been cloned and identified, all four

of the other VDAC isoforms from Arabidopsis were tested in

yeast two-hybrid experiments, and only VDAC3was identified as

interacting with KP1 (Figures 3A and 3B). To provide biochemical

evidence of this interaction, the VDAC3 and KP1 recombinant

Figure 7. KP1 Expression Increased during Seed Germination.

Real-time PCR analyses of KP1 expression in the wild type during seed imbibition and germination at 22 (A) or 48C (B). KP1 transcripts exhibit a sudden

increase during this period. The 18S rRNA gene was amplified as an internal reference. Data represent the mean 6 SE of three independent biological

determinations.

Kinesin and VDAC in Seed Respiration 1099

proteins were bacterially expressed, affinity purified, and ana-

lyzed by GST pull down and far-protein gel blot, respectively

(Figures 3C to 3E). For the direct observation of the interaction in

vivo, both BiFC and LCI assays were performed (Figures 4A and

4D). The results from these methods confirmed the specific

interaction between KP1 and VDAC3.

VDACs are the most abundant integral membrane proteins in

the mitochondrial outer membrane (Colombini, 1979; Benz,

1994). Dozens of cytosolic proteins have been reported to

interact with VDACs in mammalian and yeast cells (reviewed in

Rostovtseva and Bezrukov, 2008). Through affinity chromatog-

raphy analysis, VDAC was identified as a binding site for micro-

tubule-associated protein 2 (Linden andKarlsson, 1996). Purified

VDAC1 interacts with a cytoplasmic dynein light chain Tctex-1 in

vitro (Schwarzer et al., 2002). Recently, VDAC proteins were

shown to interact with tubulin and actin based on immunopre-

cipitation and surface plasmon resonance technology (Carre

et al., 2002; Roman et al., 2006). Interestingly, several kinesin

receptors were identified as binding to mitochondria, such as

syntabulin, kinectin, and milton (Stowers et al., 2002; Santama

et al., 2004; Cai et al., 2005). In plant cells, the relationship

between kinesin and mitochondrial proteins has not been re-

ported. The specific interaction between KP1 and VDAC3

reported here reveals novel roles for plant kinesin isoforms and

provides insight into the molecular mechanisms underlying mi-

tochondrial functions.

Although the transgenic cellular imaging study (Figure 1)

suggests that KP1 is able to target to mitochondria via its tail

domain, we remain puzzled about the regulation of the tail-cargo

binding. The binding affinity of full-length KP1 to microtubules

and mitochondria seems to be related to the regulation of the

KP1 protein domains. In Arabidopsis plants, KP1 is usually

expressed at a very low level and tightly localizes tomitochondria

(Ni et al., 2005). When transiently overexpressed in protoplasts,

GFP-KP1 fusion proteins accumulated on microtubules and,

to a small degree, on mitochondria (Figure 1C), whereas tail-

GFP preferentially localized to mitochondria (Figure 1C), sug-

gesting that the other domains of KP1 may negatively regulate

the tail-mitochondria association. Interactions between differ-

ent domains are abundant in animal kinesins. For example,

there is a direct interaction between the kinesin-1 head and

tail (Dietrich et al., 2008); a 65–amino acid C-terminal tail

domain is an inhibitory regulator of the ATPase and motor

activities of the head domains of the kinesin (Coy et al., 1999); in

the absence of bound cargo, the kinesin tail interacts with the

motor domains and inhibits their activity (Cross and Scholey,

1999). It will be interesting to establish how the head and

motor domains of KP1 influence the ability of its tail to bind to

mitochondria.

Figure 9. The kp1 and vdac3Mutants Have a Higher Aerobic Respiration

Rate during Seed Germination at 48C.

Oxygen consumption was measured in kp1 and vdac3 mutants during

the period of seed germination at 48C by a Clark-type electrode. Each

value is the mean 6 SE of three independent biological determinations.

Asterisks indicate a significant difference between mutants and Col: *P <

0.05; **P < 0.01. DW, dry weight.

Figure 8. KP1 and VDAC3 Are Involved in Seed Germination at 48C.

(A) One-month-ripened seeds were germinated at 48C with a light period

of 16 h. The KP1 dominant-negative mutants, TAILOE, and vdac3 T-DNA

insertion lines exhibited a higher seed germination frequency than the

wild type (Col) at 48C. Data points represent the mean 6 SE of three

independent biological determinations.

(B) The visually distinguishable phenotypes of the wild type (Col),

mutants, and transgenic plants at day 15 after imbibition at 48C. Bars =

2 mm.

(C) The root length of different mutant lines at day 15 after imbibition at

48C. Each value is the mean 6 SE of five independent biological

determinations. Asterisks indicate a significant difference between mu-

tants and Col: *P < 0.05; **P < 0.01.

1100 The Plant Cell

KP1 and VDAC3 Participate in Mitochondrial Respiratory

Regulation during Seed Germination

Kinesins have many cellular functions, including the transport of

organelles (Hirokawa et al., 2009). The kinesin members, KIF1B

and KIF5B in mouse cells (Nangaku et al., 1994; Tanaka et al.,

1998) and KLP67A in early Drosophila embryos (Tanaka et al.,

1998), have been implicated inmitochondrial movement. Purified

KIF1B could drive mitochondria to move along microtubules to

the plus end (Nangaku et al., 1994). It is unclear if any kinesins are

involved in mitochondrial respiration. In this research, though no

significant seed germination phenotype was observed in kp1

T-DNA insertion lines, KP1 dominant-negative mutants, TAILOE,

exhibited a higher seed germination frequency at 48C (Figure 8A),

indicating that KP1 functions as a regulator during seed germi-

nation. Further analysis with specific respiration inhibitors

(SHAM and NaN3) showed that SHAM-resistant respiration (sim-

ilar to the cytochrome pathway) significantly increased in kp1

T-DNA insertion mutants and TAILOE lines at day 10 after

imbibition, while CN-resistant respiration decreased (Figures

10A and 12A). According to previous studies on several different

plants, CN-resistant respiration predominates during the early

stages of seed germination, but after 12 h of imbibition, SHAM-

and CN-resistant respiration achieve a balance (Yentur and

Leopold, 1976). Our data showed that at day 10 after imbibition,

the balance between the SHAM- and CN-resistant respiration

pathway was disrupted in kp1 T-DNA insertion mutants and

TAILOE lines, changing from a ratio of 1:1 in the wild type to 6:1 in

mutants (Figures 10A and 12A). This suggests that KP1 regulates

respiratory pathways during seed germination.

In animal cells, the mitochondrial channel VDACs form large

aqueous pores through membranes and play a crucial role in

regulating the transport of ATP/ADP, Ca2+, and other metabo-

lites between the cytosol and mitochondria (Rostovtseva and

Colombini, 1996; Lemasters and Holmuhamedov, 2006). The

Figure 10. The Balance between the SHAM- and CN-Resistant Respi-

ration Pathways in kp1 and vdac3 Mutants Is Disrupted.

(A) The oxygen consumption of the SHAM- and CN-resistant respiration

pathways (n = 3) in Col, kp1-1, kp1-2, vdac3-1, and VOE was measured.

The test materials grown at 48C for 10 d were treated with specific

inhibitors: SHAM (15 mM) for cytochrome respiration and NaN3 (1 mM)

for AOX respiration. The balance between SHAM- and CN-resistant

respiration was disturbed in kp1 and vdac3 mutants, and the VDAC3

overexpression line (VOE) was able to restore the ratio of SHAM- to CN-

resistant respiration in vdac3-1. Each value is the mean 6 SE of three

independent biological determinations. Asterisks indicate a significant

difference between mutants and Col: *P < 0.05; **P < 0.01. DW, dry

weight.

(B) The graph represents the transgenic constructs overexpressing

VDAC3. A flag tag was fused to the N terminus of VDAC3.

(C) Immunoblot identification of flag-VDAC3 in Col and VOE using an

anti-flag antibody (1:10,000). Molecular markers are indicated in kilo-

daltons in the right margin.

Figure 11. ATP Measurements in kp1 and vdac3 Mutants Grown at 48C.

(A) Ten-day-old seedlings were used for ATP quantification by the

luciferin-luciferase method. The ATP levels are lower in kp1-1, kp1-2, and

vdac3-1 and are restored in VOE. Each value is the mean 6 SE of three

independent biological determinations. Asterisks indicate significant

differences between mutants and Col: *P < 0.05; **P < 0.01. FW, fresh

weight.

(B)Citrate synthase activities of 10-d-old Col, kp1-1, kp1-2, vdac3-1, and

VOE grown at 48C were assayed to eliminate the possibility of different

mitochondrial numbers in the different lines examined. No significant

differences were observed. Each value is the mean 6 SE of three

independent biological determinations.

Kinesin and VDAC in Seed Respiration 1101

reduction in the expression of VDAC1 in human cells leads to a

decrease in the ATP level (Abu-Hamad et al., 2006). In this study,

the vdac3mutants shared a similar phenotype to KP1 dominant-

negative mutants, TAILOEs, and kp1mutants, including a higher

cold germination frequency (Figure 8A), a higher respiration rate

(Figure 9), a higher ratio of SHAM- to CN-resistant respiration

(;3:1) (Figure 10A), and lower ATP levels (Figure 11A). This

indicates that VDAC3 also functions in regulating respiratory

pathways during seed germination at low temperature. Recently,

it was reported that in Trypanosoma brucei, a unicellular parasitic

protozoan, the downregulation of Tb VDAC reduces cellular

respiration via the AOX pathway, enhances respiration via the

cytochrome pathway, and exhibits a lower ATP level (Singha

et al., 2009).

What is the mechanism by which KP1 interacts with VDAC3?

What is the relationship between respiration pathway balance

and seed germination in both kp1 and vdac3 mutants at low

temperature? From our data, we could not answer these ques-

tions. We suggest that KP1 may positively regulate the opening

of VDAC3 and regulate the flux of ATP/ADP across the mito-

chondrial outer membrane; in mutants, the ATP/ADP flux may

be disturbed to a certain extent, resulting in a lower production

of ATP in mitochondria, which in turn stimulates respiration via

the cytochrome pathway in an attempt to compensate for the

energy crisis in the cells. It would be interesting to address the

above questions.

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana seeds were surface terilized. The seeds were strat-

ified at 48C for 2 d and then placed on half-strengthMurashige and Skoog

(MS)medium (1%Suc and 0.9%agar). For the transformation andmutant

screen, seedlings were transferred from plates to soil and grown at 228C

in a growth room with a photon flux density of 100 mEm22 s21 and a 16-h

photoperiod.

For the seed germination assays, seeds from the different genotypes

were harvested from plants grown simultaneously in glasshouse condi-

tions and then stored for 4 weeks. For the seed germination assay at 48C,

plates were kept in the incubator at 48C with a 16-h photoperiod at a

photon flux density of 70 mE/m22 s21. Germination was scored by radicle

emergence.

Plasmid Construction

The full-length KP1 cDNA was previously cloned (Li et al., 2007). The full-

length cDNA sequence of VDAC3 is available from GenBank (accession

number NM_121513). For protoplast transient expression assays, the full-

length cDNA of KP1 was amplified by PCR using the following two

primers: 59-TCTAGAATGGACCAAGGCGCGAT-39 (XbaI) and 59-GTC-

GACCTATGGTACCATGAACCTTG-39 (SalI). The KP1-Dtail (KP1 lacking

the C-terminal region; 1 to 751 amino acids) construct was made using

the following primers: 59-TCTAGAATGGACCAAGGCGCGAT-39 (XbaI)

and 59-GTCGACCTAGTTCCGAATGCTACCT-39 (SalI). For the KP1-tail

(749 to 1087 amino acids), the primers 59-TCTAGAATGAAGGAAA-

CCGGTGAAATTC-39 (XbaI) and 59-GTCGACTGGTACCATGAACCT-

TGC-39 (SalI) were used. For VDAC3, the primers 59-TCTAGAA-

TGGTTAAAGGTCCAGGACTCTAC-39 (XbaI) and 59-GAGCTCTCAGGG-

CTTGAGAGCGAGAG-39 (SacI) were used. The open reading frames

(ORFs) of GFP were amplified using the following primers: 59-GGATC-

CATGAGTAAAGGAGAAGAACT-39 (BamHI) and 59- TCTAGATTTGTA-

TAGTTCATCCATGCC-39 (XbaI) with no termination codon, and 59-GTC-

GACATGAGTAAAGGAGAAGAAC-39 (SalI) and 59-TTAGGTACCTTTG-

TATAGTTCATCCATG-39 (SacI) with the termination codon TAA. The

fusion constructs GFP-KP1, GFP-Dtail, and GFP-VDAC3 were made by

placing the coding region in frame with the C terminus of the GFP coding

region (digested with BamHI and XbaI). The tail-GFP construct was made

by fusing the target sequences to the N terminus of GFP (digested with

SalI and SacI). All of the fusion constructs above were cloned into a pUC

vector under the control of the 35S cauliflower mosaic virus promoter.

For the BiFC assay, the ORFs of VDAC3 were amplified by PCR using

the following primers: 59-TCTAGAATGGTTAAAGGTCCAGGACTCTAC-39

(XbaI) and 59-CTCGAGGGGCTTGAGAGCGAGAG-39 (XhoI). The DNA

fragments encoding full-length KP1 were amplified by PCR using the

following primers: 59-TCTAGAATGGACCAAGGCGCGAT-39 (XbaI) and

59-GTCGACTGGTACCATGAACCTTGC-39 (SalI). All of the DNA frag-

ments used were cloned into the plasmid pUC-SPYNE to form the fusion

proteins KP1-YFPN, KP1-tail-YFPN, and VDAC3-YFPN and into pUC-

SPYCE to form the fusion proteins KP1-YFPC, KP1-tail-YFPC, and

VDAC3-YFPC. For fusing YFPC to the N terminus of VDAC3, we amplified

Figure 12. The Ratio of SHAM- to CN-Resistant Respiration and ATP

Levels in TAILOE Dominant-Negative Mutants.

(A) The oxygen consumption of the SHAM/CN-resistant respiration

pathways (n = 3) in Col, TAILOE1, and TAILOE2 was measured. The

balance between SHAM/CN-resistant respiration is disturbed in TAILOE

lines. DW, dry weight.

(B) The ATP levels are lower in TAILOE lines. Each value is the mean6 SE

of three independent biological determinations. Asterisks indicate sig-

nificant differences between mutants and Col: *P < 0.05; **P < 0.01. FW,

fresh weight.

1102 The Plant Cell

the C-terminal half of YFP by PCR using the following primers:

59-TCTAGAATGTACGACGTACCAGATTA-39 (XbaI) and 59-ACTAGT-

CTTGTACAGCTCGTCCAT-39 (SpeI). We amplified the ORFs of VDAC3

using the following primers: 59-ACTAGTCGATCACTACAACGGGAAC-39

(SpeI) and 59-TCTAGAAGAAGAATCAGTGGAAACTTTG-39 (XbaI) with a

termination codon. TheseDNA fragments were inserted into pUC-SPYCE

to form YFPC-VDAC3. All of the constructs above were sequenced to

confirm their fidelity.

Screening of T-DNA Insertion Lines and Isolation of kp1 and

vdac3Mutants

The kp1-1 (SALK_056981), kp1-2 (SALK_117309), vdac3-1 (SALK_

127899), and vdac3-2 (SAIL_238_A01) mutant lines, carrying T-DNA

insertions in KP1 and VDAC3, were obtained from the ABRC. PCR-based

screening was used to test homozygosity. The locations of the T-DNA

insertion sites in the SALK and SAIL lines were determined by direct

sequencing of PCR products amplified by the T-DNA left border primer

LBa1, 59-TGGTTCACGTAGTGGGCCATCG-39 for SALK lines, the primer

LB3, 59-TAGCATCTGAATTTCATAACCAATCTCGATACAC-39 for SAIL

lines, and gene-specific primers. The gene-specific primers were as fol-

lows: for kp1-1 (SALK_056981), 59-CACACAGCACCATAAGCATTG-39;

for kp1-2 (SALK_117309), 59-GAAGCAGAGCTGGAACAATTG-39; for

vdac3-1 (SALK_127988), 59-AGACATTGTCAAAGACTCAACAAC-39; and

for vdac3-2 (SAIL_238_A01), 59-TGCCAGATTCGGTGTTATAGG-39.

Generation of Transgenic Plants

The plasmid constructs pBI121-35S--flag-VDAC3 and pBI121-35S-KP1-

tail-GFP were electroporated into Agrobacterium tumefaciens strain

C58C1. Stable transgenic Arabidopsis plants were generated using the

flower dipping method (Clough and Bent, 1998). Transgenic plants were

selected on 0.53MSplates with 50mg/L kanamycin. The T3 generations

of transgenic plants were used for experiments.

RT-PCR and Real-Time PCR

Total RNA was isolated from plant materials using Trizol reagent (Invi-

trogen) according to the manufacturer’s instructions. RNA isolation from

the dry, imbibed, and germinating seeds was performed using the RNA

extraction kit (Bioteke) according to themanufacturer’s instructions. RNA

concentration was determined using a NanoDrop ND-1000 photospec-

trometer. Reverse transcription was performed with 5 mg of total RNA

using M-MLV reverse transcriptase (Promega) according to the manu-

facturer’s instructions. PCR was performed for 35 cycles with the follow-

ing gene-specific primers: UBQ5, 59-CTCCTTCTTTCTGGTAAACGT-39

and 59-GGTGCTAAGAAGAGGAAGAAT-39; KP1, 59-CAGAAGCTACGA-

GACCAGAAGTTG-39 and 59-CTATGGTACCATGAACCTTGCATG-39;

and VDAC3, 59-TTTTTCCAGAGGCAATCATG-39 and 59-GCCCATTTGG-

TGGTATCTTC-39. Quantitative real-time PCR was performed following

the protocol of the Perfect Real-time PCR kit (TaKaRa) on the Applied

Biosystems 7500 Real-Time PCR system. Amplification products were

visualized by SYBR Green. Aliquots of the RT reaction products were

used as templates for real-time PCR reactions. For relative quantification,

the 18S rRNA gene was detected as an internal reference, and the 22DDCt

method (Livak and Schmittgen, 2001) was used.

Protoplast Transient Expression Assays and

Fluorescence Microscopy

Transient expression of various plasmids in Arabidopsis protoplasts was

performed as previously described (Sheen, 2002). The viability of proto-

plasts was determined using fluorescein diacetate staining (Larkin, 1976)

(see Supplemental Figure 3 online). For GFP-KP1, both bright and dim

protoplasts were chosen for observation. The BiFC assay was performed

according to a previous report (Walter et al., 2004). Fluorescence was

observed after protoplasts were incubated at 228C for 12 to 16 h.

Mitochondria were visualized by staining with MitoTracker Red (Molec-

ular Probes) according to the manufacturer’s protocol. To stain microtu-

bules and microfilaments, Arabidopsis suspension cell protoplasts were

attached to cover slips coated with 1 mg/mL poly-L-lysine (Mr >300,000;

Sigma-Aldrich) and fixed for 30 min at room temperature with 3% (w/v)

paraformaldehyde in PEMbuffer (50mMPIPES, pH 6.9, 5mMEGTA, and

1 mMMgSO4) supplemented with 1%DMSO, 0.3 mM PMSF, and 0.05%

Triton X-100. The fixed protoplast ghosts were thenwashedwith PBS, pH

7.4, and blocked in 1% (w/v) BSA (Sigma-Aldrich) for 10 min. For labeling

microtubules, the anti-a-tubulin monoclonal antibody (Sigma-Aldrich;

diluted 1:500) and the tetramethylrhodamine b-isothiocyanate–conju-

gated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories;

diluted 1:200) were applied, and microfilaments were stained with 50 nM

rhodamine-phalloidin (Molecular Probes) at 258C for 1 h. After rinsing in

PBS, slides were observed under a Zeiss LSM 510 META confocal

microscope. The confocal settings were as follows: green images (GFP,

fluorescein diacetate staining) were obtained with a 488-nm argon laser

and Fset09 wf filter; red images (MitoTracker stain, rhodamine-phalloidin

stain) were obtained with a 543-nm HeNe laser and Fset15 wf filter. A

photomultiplier tube was used as the confocal detector.

Protein Expression and Antibody Production

His-VDAC3 proteins were expressed in the Escherichia coli BL21 (DE3)

strain, induced with 0.1 mM isopropyl thiogalactoside for 6 h at 228C, and

affinity purified using a Ni2+-chelating Sepharose Fast Flow (Amersham

Biosciences) column following the manufacturer’s instructions.

Polyclonal anti-VDAC3 antibodies were raised in rabbits using the

purified His-VDAC3 protein as the antigen. Antiserum was then affinity

purified using the AminoLink Plus kit (Pierce Chemical) with immobilized

VDAC3 according to the manufacturer’s instructions.

Yeast Two-Hybrid Screening

Arabidopsis cDNA clones encoding KP1-interacting proteins were

screened by a GAL4-based yeast two-hybrid system using the yeast

two-hybrid host strain AH109 as described by the manufacturer (Clon-

tech). KP1-tail was used in the bait construct to screen the cDNA library

for candidate interaction partners of KP1. To verify the interaction

between KP1 and VDAC3, truncated KP1 proteins, KP1-N (1 to 373

amino acids), KP1-M (374 to 749 amino acids), and KP1-tail200 (888 to

1087 amino acids) were used for bait constructs.

For the interactions between KP1 and VDAC family proteins, the ORF

cDNA of VDAC1 was amplified by PCR using the following two primers:

59-CCTCCAACTTTCTCAGATAAGCAAC-39 and 59-CTGAATATGCAA-

TTTTCATTATGACAC-39. For VDAC2, 59-CTCTCTCAATCTCCGATCA-

ACC-39 and 59-CTGCGGAACTATTTATTGATTCC-39 were used, and

for VDAC4, 59-GCATTTGTTTTCTATATCCGAAG-39 and 59-TCCCTTT-

TCTTTCACATCACA-39 were used. The full-length cDNAs of VDAC1,

VDAC2, and VDAC4 were then ligated into pGADT7 and transformed into

yeast cells with pGBKT7-KP1-tail and pGBKT7-KP1-tail200.

GST Pull Down and Far-Protein Blot Assays

GST, GST-VDAC3, His-VDAC3, and His-KP1-tail200 were expressed in E.

coli BL21 (DE3) and purified according to standard protocols (Sambrook

and Russell, 2001).

A GST pull-down assay was conducted. Aliquots of GST and GST-

VDAC3 beads (100 mL beads containing ;15 mg of protein) were

incubated for 2 h at 48C with the His-KP1-tail200 protein. After being

washed with PBS buffer, bound proteins were eluted from the beads with

Kinesin and VDAC in Seed Respiration 1103

50 mL of elution buffer (20 mM reduced glutathione in 50 mM Tris-Cl, pH

8.0), resolved on a 12.5% SDS-PAGE gel, and then immunoblotted with

anti-His antibody at a 1:10,000 dilution and with anti-GST antibody at a

1:100,000 dilution.

A far-protein gel blot was performed as previously described

(Schwarzer et al., 2002). The total proteins extracted from E. coli over-

expressing His-VDAC3 and GST-VDAC3 and the purified His-VDAC3,

GST-VDAC3, His-KP1-tail200, and unrelated His-tagged recombinant

protein were separated by SDS-PAGE and transferred electrophoretically

to PVDF membranes. After the blocking of nonspecific sites with 5%

nonfat dried milk in TBST (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and

0.05% Tween) for 2 h at room temperature, the membranes were washed

three times with TBST and incubated for 1 h at room temperature at 48C

with the recombinant His-KP1-tail200 protein (20mg/mL). Themembranes

were then washed three times in TBST and blocked again. Then, the

membranes were immunoblotted with anti-His, anti-GST, anti-KP1, and

anti-VDAC3 antibody, respectively.

LCI Assay

The LCI assay was performed as previously described (Chen et al., 2008).

To make KP1-/KP1-tail-NLuc, VDAC3-NLuc, CLuc-KP1/KP1-tail, and

CLuc-VDAC3 constructs for the LCI assay, the related cDNAs were

inserted into pCAMBIA-NLuc and pCAMBIA-CLuc vectors. All of the

resultant constructs were electroporated into Agrobacterium strain

C58C1. Bacterial suspensions were infiltrated into fully expanded leaves

of the 7-week-old Nicotiana benthamiana plants using a needleless

syringe. After that, plants were grown in darkness for 12 h and then with a

light period of 16 h for 60 h at 228C. The LUC activity was observed with a

CCD imaging apparatus (AndoriXon; Andor).

Measurements of Oxygen Consumption

Seeds (50 mg) germinated at 48C in liquid culture medium (0.53 MS

medium, 0.025% MES, and 1% Suc, pH 5.6) were used to measure

oxygen consumption at 48C using 1.5 mL medium in the dark, a Clark-

type electrode (Hansatech), and a 2-mL vessel. The oxygen consumption

in the seeds (germinated for 10 d) was detected in the presence or

absence of the specific respiratory inhibitors, 1 mM NaN3 and 15 mM

SHAM.

Measurements of ATP Level and Citrate Synthase Activity

The ATP concentration was measured as previously described (Meyer

et al., 2009). To extract ATP from the seedlings, ;250 mg frozen

Arabidopsis seedlings were ground and resuspended in 400 mL of

2.3% (v/v) trichloroacetic acid. A bioluminescent assay kit (Sigma-

Aldrich) was used to measure the ATP concentration. Citrate synthase

activity was assayed spectrophotometrically at 412 nm in a reaction with

0.2mMoxaloacetate, 0.1mMacetyl-CoA, and 0.2mM5, 59-dithiobis(2,4-

nitrobenzoic acid) (Bond et al., 2005). The change of OD in a unit

represents a U-activity unit.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession

numbers: KP1 (At3g44730), VDAC1 (At3g01280), VDAC2 (At5g67500),

VDAC3 (At5g15090), VDAC4 (At5g57490), and UBQ5 (At3g62250).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. GFP-KP1 Decorated Microtubules in Arabi-

dopsis Suspension Cell Protoplasts.

Supplemental Figure 2. There Is No Significant Difference in Mito-

chondrial Organization and Morphology in Roots among 5-d-Old Col

and kp1-1 and vdac3-1 Mutants.

Supplemental Figure 3. The Viability of Protoplasts Was Determined

Using Fluorescein Diacetate.

ACKNOWLEDGMENTS

We thank Shao-Hua Li (Institute of Botany, Chinese Academy of Sci-

ences) for providing a Clark-type electrode for the measurement of

oxygen consumption. This work was supported by grants from the

National Natural Science Foundation of China (Project 31071259,

30770128, 30721062, and 31030010).

Received December 18, 2010; revised February 10, 2011; accepted

February 21, 2011; published March 15, 2011.

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1106 The Plant Cell

DOI 10.1105/tpc.110.082420; originally published online March 15, 2011; 2011;23;1093-1106Plant CellGuo-Qin Liu

Xue-Yong Yang, Zi-Wei Chen, Tao Xu, Zhe Qu, Xiao-Di Pan, Xing-Hua Qin, Dong-Tao Ren andRegulates Respiration during Seed Germination at Low Temperature

Kinesin KP1 Specifically Interacts with VDAC3, a Mitochondrial Protein, andArabidopsis

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