|Arabidopsis Kinesin KP1 Specifically Interacts with VDAC3, a Mitochondrial Protein, and Regulates Respiration during Seed Germination at Low Temperature.|
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|PMID: 21406623 Owner: NLM Status: Publisher|
|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 mitochondrial 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 4°C, KP1 dominant-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.|
|Xue-Yong Yang; Zi-Wei Chen; Tao Xu; Zhe Qu; Xiao-Di Pan; Xing-Hua Qin; Dong-Tao Ren; Guo-Qin Liu|
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|Type: JOURNAL ARTICLE Date: 2011-3-15|
|Title: The Plant cell Volume: - ISSN: 1532-298X ISO Abbreviation: - Publication Date: 2011 Mar|
|Created Date: 2011-3-16 Completed Date: - Revised Date: -|
Medline Journal Info:
|Nlm Unique ID: 9208688 Medline TA: Plant Cell Country: -|
|Languages: ENG Pagination: - Citation Subset: -|
|State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.|
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Journal ID (nlm-ta): Plant Cell
Journal ID (hwp): plantcell
Journal ID (publisher-id): aspb
Publisher: American Society of Plant Biologists
© 2011 American Society of Plant Biologists
Received Day: 18 Month: 12 Year: 2010
Revision Received Day: 10 Month: 2 Year: 2011
Accepted Day: 21 Month: 2 Year: 2011
Print publication date: Month: 3 Year: 2011
Electronic publication date: Day: 15 Month: 3 Year: 2011
pmc-release publication date: Day: 15 Month: 3 Year: 2011
Volume: 23 Issue: 3
First Page: 1093 Last Page: 1106
PubMed Id: 21406623
Publisher Id: 082420
|Arabidopsis Kinesin KP1 Specifically Interacts with VDAC3, a Mitochondrial Protein, and Regulates Respiration during Seed Germination at Low Temperature[W][OA]|
|State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China
|3Address correspondence to email@example.com.
1These authors contributed equally to this work.
2Guo-Qin Liu and Dong-Tao Ren's groups contributed equally to this work.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Guo-Qin Liu (firstname.lastname@example.org).
[W]Online version contains Web-only data.
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Much of the aerobic oxidation in eukaryotic cells takes place in mitochondria. A number of studies have shown that microfilaments and microtubules function in mitochondrial movement and positioning in eukaryotic cells (Hirokawa, 1998). Cytoskeletal 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 dependent 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 (Carré 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 metabolites, 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 techniques, 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 transduction (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 transient 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 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 presence 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 interaction 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 (4°C).
According to our previous work, KP1 is found in isolated mitochondria (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 mechanism underlying the interaction between KP1 and mitochondria, GFP-KP1 and its truncated proteins, Δtail (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 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 mitochondria (Figure 1C, white arrows). Interestingly, tail-GFP was located in mitochondria, but GFP-Δtail 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 (Figure 2A) and transformed into Arabidopsis. Two overexpression lines (TAILOE1 and TAILOE2) were identified by immunoblotting with a monoclonal antibody against GFP (Figure 2B). The results indicate that tail-GFP indeed localizes to mitochondria (Figure 2C).
To identify if the tail of KP1 interacts with mitochondrial proteins, we performed a yeast two-hybrid screen using the tail polypeptide 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 domains 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 vitro was verified by a GST pull 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 polyvinylidene difluoride (PVDF) membranes. After the membranes were incubated with 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 3′; far-western, lane 3′) 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- tail200 in vitro.
The specific interaction in vivo between KP1 and VDAC3 was verified by two methods. Bimolecular fluorescence complementation (BiFC) showed that both KP1 and KP1-tail interact with VDAC3 (Figure 4A). The interaction occurs specifically at mitochondria (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 (Nicotiana tabacum) leaf cells, fluorescence signals were detected (Figure 4D). All of these results demonstrate that KP1 specifically interacts 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 kp1 mutants, the T-DNA inserted within the exon; in vdac3-1, the T-DNA inserted in the 5′-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 expression of KP1 was completely interrupted by the T-DNA insertion in the kp1 mutants, and VDAC3 expression was significantly reduced in vdac3 mutants (Figure 5D).
We then tested the localization of tail-GFP in vdac3-1 mutants 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 colocalized with MitoTracker Red, suggesting that the mitochondrial localization of KP1 is dependent on its interaction with VDAC3.
The phenotypes of all kp1 mutants and transgenic plants prepared in this study were examined under normal growth conditions 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 4°C, 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 treatment. Interestingly, vdac3 T-DNA insertion lines exhibited a higher seed germination frequency than the wild type at 4°C, 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 4°C, 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 redundant proteins in plant cells. As expected, the transgenic lines showed similar phenotypes to the vdac3 mutants at 4°C (Figures 8A to 8C).
To determine if the visual phenotype of the mutants at 4°C was due to quicker germination but not faster growth, seeds were germinated at 22°C for 2 d before being transferred to 4°C. The mutants did not present any obvious morphological phenotype. These results demonstrate that both KP1 and VDAC3 function during seed germination at a low temperature.
The germination of seeds at the dormant stage requires efficient energy from degradation metabolism. In a dry seed, the 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 respiratory rate of kp1 and vdac3 mutants was investigated. As shown in Figure 9, oxygen consumption increased significantly in kp1 and vdac3 mutants during the period of seed germination at 4°C, 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 4°C in the presence of the specific respiration inhibitors, NaN3 (for inhibiting the cytochrome pathway) and salicylhydroxamic 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 overexpression 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 cytochrome pathway is mainly used for ATP synthesis. Therefore, we measured the ATP levels in 10-d-old seedlings. Interestingly, the kp1 mutants grown at 4°C 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 mutant phenotype. To eliminate the possibility that the observed changes in ATP levels were due to a different quantity of mitochondria, the activity of citrate synthase, a marker for mitochondrial 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 involved in regulating respiration pathways and ATP levels during seed germination at a low temperature.
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 in microtubule organization during meiosis 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.
Systematic analysis of the sequences in the Arabidopsis 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 membrane 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 VDAC3 was identified as interacting with KP1 (Figures 3A and 3B). To provide biochemical evidence of this interaction, the VDAC3 and KP1 recombinant proteins were bacterially expressed, affinity purified, and analyzed 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 chromatography analysis, VDAC was identified as a binding site for microtubule-associated protein 2 (Lindén and Karlsson, 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 immunoprecipitation and surface plasmon resonance technology (Carré 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 reported. The specific interaction between KP1 and VDAC3 reported here reveals novel roles for plant kinesin isoforms and provides insight into the molecular mechanisms underlying mitochondrial 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 to mitochondria (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), suggesting that the other domains of KP1 may negatively regulate the tail-mitochondria association. Interactions between different 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.
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 in mitochondrial 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 4°C (Figure 8A), indicating that KP1 functions as a regulator during seed germination. Further analysis with specific respiration inhibitors (SHAM and NaN3) showed that SHAM-resistant respiration (similar 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 metabolites between the cytosol and mitochondria (Rostovtseva and Colombini, 1996; Lemasters and Holmuhamedov, 2006). The 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 vdac3 mutants shared a similar phenotype to KP1 dominant-negative mutants, TAILOEs, and kp1 mutants, 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 questions. We suggest that KP1 may positively regulate the opening of VDAC3 and regulate the flux of ATP/ADP across the mitochondrial 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.
Arabidopsis thaliana seeds were surface terilized. The seeds were stratified at 4°C for 2 d and then placed on half-strength Murashige and Skoog (MS) medium (1% Suc and 0.9% agar). For the transformation and mutant screen, seedlings were transferred from plates to soil and grown at 22°C in a growth room with a photon flux density of 100 μE m−2 s−1 and a 16-h photoperiod.
For the seed germination assays, seeds from the different genotypes were harvested from plants grown simultaneously in glasshouse conditions and then stored for 4 weeks. For the seed germination assay at 4°C, plates were kept in the incubator at 4°C with a 16-h photoperiod at a photon flux density of 70 μE/m−2 s−1. Germination was scored by radicle emergence.
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: 5′-TCTAGAATGGACCAAGGCGCGAT-3′ (XbaI) and 5′-GTCGACCTATGGTACCATGAACCTTG-3′ (SalI). The KP1-Δtail (KP1 lacking the C-terminal region; 1 to 751 amino acids) construct was made using the following primers: 5′-TCTAGAATGGACCAAGGCGCGAT-3′ (XbaI) and 5′-GTCGACCTAGTTCCGAATGCTACCT-3′ (SalI). For the KP1-tail (749 to 1087 amino acids), the primers 5′-TCTAGAATGAAGGAAACCGGTGAAATTC-3′ (XbaI) and 5′-GTCGACTGGTACCATGAACCTTGC-3′ (SalI) were used. For VDAC3, the primers 5′-TCTAGAATGGTTAAAGGTCCAGGACTCTAC-3′ (XbaI) and 5′-GAGCTCTCAGGGCTTGAGAGCGAGAG-3′ (SacI) were used. The open reading frames (ORFs) of GFP were amplified using the following primers: 5′-GGATCCATGAGTAAAGGAGAAGAACT-3′ (BamHI) and 5′- TCTAGATTTGTATAGTTCATCCATGCC-3′ (XbaI) with no termination codon, and 5′-GTCGACATGAGTAAAGGAGAAGAAC-3′ (SalI) and 5′-TTAGGTACCTTTGTATAGTTCATCCATG-3′ (SacI) with the termination codon TAA. The fusion constructs GFP-KP1, GFP-Δtail, 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: 5′-TCTAGAATGGTTAAAGGTCCAGGACTCTAC-3′ (XbaI) and 5′-CTCGAGGGGCTTGAGAGCGAGAG-3′ (XhoI). The DNA fragments encoding full-length KP1 were amplified by PCR using the following primers: 5′-TCTAGAATGGACCAAGGCGCGAT-3′ (XbaI) and 5′-GTCGACTGGTACCATGAACCTTGC-3′ (SalI). All of the DNA fragments 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 the C-terminal half of YFP by PCR using the following primers: 5′-TCTAGAATGTACGACGTACCAGATTA-3′ (XbaI) and 5′-ACTAGTCTTGTACAGCTCGTCCAT-3′ (SpeI). We amplified the ORFs of VDAC3 using the following primers: 5′-ACTAGTCGATCACTACAACGGGAAC-3′ (SpeI) and 5′-TCTAGAAGAAGAATCAGTGGAAACTTTG-3′ (XbaI) with a termination codon. These DNA fragments were inserted into pUC-SPYCE to form YFPC-VDAC3. All of the constructs above were sequenced to confirm their fidelity.
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, 5′-TGGTTCACGTAGTGGGCCATCG-3′ for SALK lines, the primer LB3, 5′-TAGCATCTGAATTTCATAACCAATCTCGATACAC-3′ for SAIL lines, and gene-specific primers. The gene-specific primers were as follows: for kp1-1 (SALK_056981), 5′-CACACAGCACCATAAGCATTG-3′; for kp1-2 (SALK_117309), 5′-GAAGCAGAGCTGGAACAATTG-3′; for vdac3-1 (SALK_127988), 5′-AGACATTGTCAAAGACTCAACAAC-3′; and for vdac3-2 (SAIL_238_A01), 5′-TGCCAGATTCGGTGTTATAGG-3′.
The plasmid constructs pBI121-35S-Ω-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 0.5× MS plates with 50 mg/L kanamycin. The T3 generations of transgenic plants were used for experiments.
Total RNA was isolated from plant materials using Trizol reagent (Invitrogen) 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 the manufacturer’s instructions. RNA concentration was determined using a NanoDrop ND-1000 photospectrometer. Reverse transcription was performed with 5 μg of total RNA using M-MLV reverse transcriptase (Promega) according to the manufacturer’s instructions. PCR was performed for 35 cycles with the following gene-specific primers: UBQ5, 5′-CTCCTTCTTTCTGGTAAACGT-3′ and 5′-GGTGCTAAGAAGAGGAAGAAT-3′; KP1, 5′-CAGAAGCTACGAGACCAGAAGTTG-3′ and 5′-CTATGGTACCATGAACCTTGCATG-3′; and VDAC3, 5′-TTTTTCCAGAGGCAATCATG-3′ and 5′-GCCCATTTGGTGGTATCTTC-3′. 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 2−ΔΔCt method (Livak and Schmittgen, 2001) was used.
Transient expression of various plasmids in Arabidopsis protoplasts was performed as previously described (Sheen, 2002). The viability of protoplasts 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 22°C for 12 to 16 h. Mitochondria were visualized by staining with MitoTracker Red (Molecular Probes) according to the manufacturer’s protocol. To stain microtubules 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 PEM buffer (50 mM PIPES, pH 6.9, 5 mM EGTA, and 1 mM MgSO4) supplemented with 1% DMSO, 0.3 mM PMSF, and 0.05% Triton X-100. The fixed protoplast ghosts were then washed with PBS, pH 7.4, and blocked in 1% (w/v) BSA (Sigma-Aldrich) for 10 min. For labeling microtubules, the anti-α-tubulin monoclonal antibody (Sigma-Aldrich; diluted 1:500) and the tetramethylrhodamine β-isothiocyanate–conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories; diluted 1:200) were applied, and microfilaments were stained with 50 nM rhodamine-phalloidin (Molecular Probes) at 25°C 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.
His-VDAC3 proteins were expressed in the Escherichia coli BL21 (DE3) strain, induced with 0.1 mM isopropyl thiogalactoside for 6 h at 22°C, 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.
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 (Clontech). 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: 5′-CCTCCAACTTTCTCAGATAAGCAAC-3′ and 5′-CTGAATATGCAATTTTCATTATGACAC-3′. For VDAC2, 5′-CTCTCTCAATCTCCGATCAACC-3′ and 5′-CTGCGGAACTATTTATTGATTCC-3′ were used, and for VDAC4, 5′-GCATTTGTTTTCTATATCCGAAG-3′ and 5′-TCCCTTTTCTTTCACATCACA-3′ 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, 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 μL beads containing ~15 μg of protein) were incubated for 2 h at 4°C with the His-KP1-tail200 protein. After being washed with PBS buffer, bound proteins were eluted from the beads with 50 μL 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 overexpressing 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 4°C with the recombinant His-KP1-tail200 protein (20 μg/mL). The membranes 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.
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 22°C. The LUC activity was observed with a CCD imaging apparatus (AndoriXon; Andor).
Seeds (50 mg) germinated at 4°C in liquid culture medium (0.5× MS medium, 0.025% MES, and 1% Suc, pH 5.6) were used to measure oxygen consumption at 4°C 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.
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 0.2 mM oxaloacetate, 0.1 mM acetyl-CoA, and 0.2 mM 5, 5′-dithiobis(2,4-nitrobenzoic acid) (Bond et al., 2005). The change of OD in a unit represents a U-activity unit.
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).
The following materials are available in the online version of this article.
- Supplemental Figure 1. GFP-KP1 Decorated Microtubules in Arabidopsis Suspension Cell Protoplasts.
- Supplemental Figure 2. There Is No Significant Difference in Mitochondrial 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.
Click here for additional data file (supp_tpc.110.082420_Liu_Supplemental_Data_Final.pdf)
We thank Shao-Hua Li (Institute of Botany, Chinese Academy of Sciences) 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).
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