JXB Advance Access originally published online on February 27, 2008
Journal of Experimental Botany 2008 59(4):995-1005; doi:10.1093/jxb/ern024
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Characterization of the plant uncoupling protein, SrUCPA, expressed in spadix mitochondria of the thermogenic skunk cabbage
1Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan
2Department of Agro-Science, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
* To whom correspondence should be addressed. E-mail: ykoito{at}iwate-u.ac.jp
Received 6 December 2007; Revised 10 January 2008 Accepted 16 January 2008
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Abstract |
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In mammalian brown adipose tissue, uncoupling protein 1 (UCP1), an integral inner mitochondrial membrane protein, triggers a proton leak and converts the energy generated by the resulting electron flow into heat. Although the recent finding of plant UCPs in non-thermogenic tissues has questioned their involvement in thermogenesis, there are few studies of plant UCPs in thermogenic tissues. Therefore, in this work, two cloned UCP cDNAs, SrUCPA and SrUCPB, isolated from the thermogenic spadix of skunk cabbage, were analysed. SrUCPA, not SrUCPB, was identified as the major uncoupling protein, and it was found to be integrated into the inner mitochondrial membrane. Topological analyses indicate that the 1st and 2nd intra-matrix loops are sensitive to trypsin treatment, but the 3rd intra-matrix loop is resistant to it. Using spadix mitochondria, the uncoupling activity of SrUCPA was examined. Although SrUCPA transcripts were constitutively expressed in various tissues irrespective of thermogenic stage, the SrUCPA protein was detected only in the thermogenic tissue or stage. On the other hand, both gene and protein expression for another heat-generating protein, SrAOX, were increased specifically in the thermogenic tissue or stage. Quantitative immunoblot analysis revealed that SrUCPA was an abundant protein in spadix mitochondria, accounting for about 3% of the total mitochondrial protein in the spadix. The results suggest that specific co-expression of SrUCPA and SrAOX protein in the thermogenic tissue or stage, as well as the high expression of SrUCPA protein in spadix mitochondria, may play a role in thermogenesis of skunk cabbage.
Key words: Alternative oxidase, plant mitochondria, skunk cabbage, thermogenesis, uncoupling protein
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Introduction |
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The skunk cabbage (Symplocarpus renifolius) is a well-known thermogenic plant which grows in the north of Japan (Nie et al., 2006). In freezing temperatures, the spadix, which is the thermogenic organ of skunk cabbage, can produce enough heat to melt the ice around it (Knutson, 1974). The skunk cabbage is a protogynous plant (Seymour and Blaylock, 1999). During the female stage, which usually lasts 1–2 weeks, the spadix maintains a constant temperature around 20 °C against drastic changes in ambient temperature. On the other hand, during the male stage the spadix temperature is not regulated due to the absence of thermogenicity (Seymour and Blaylock, 1999).
Uncoupling proteins (UCP) belong to the mitochondrial anion carrier family of proteins, which are localized in the inner membrane; they partially uncouple respiration from ATP synthesis by catalysing proton leakage. All of these carriers have a molecular mass close to 33 kDa and consist of three tandemly repeated homologous domains, each with two hydrophobic stretches (Krauss et al., 2005). In cold-adapted brown adipose tissue (BAT), UCP1 levels can reach up to about 5% of total mitochondrial proteins (Ricquier et al., 1984; Pecqueur et al., 2001; Harper et al., 2002) and it plays a key role in non-shivering thermogenesis (Klingenberg, 1990). However, the recent findings that plant uncoupling proteins are expressed in non-thermogenic tissues bring into question their involvement in thermogenesis. These results suggest that plant UCPs are involved in the regulation of energy metabolism or in the reduction of reactive oxygen species in mitochondria (Brandalise et al., 2003; Considine et al., 2003; Fernie et al., 2004; Hourton-Cabassa et al., 2004). Recently it was also shown, by using a knockout mutant, that UCP1 in Arabidopsis leaves is related to photosynthetic metabolism (Sweetlove et al., 2006).
Even though much attention has been paid to UCPs in non-thermogenic plants, there has been little work with plant UCPs in thermogenic plants. Therefore, it remains to be determined whether or not thermogenic plant UCPs are involved in thermogenesis. The thermogenic spadix of skunk cabbage contains two cold-inducible cDNAs, SrUCPA and SrUCPB, encoding a canonical and a non-canonical plant UCP, respectively (Ito, 1999), and it was shown that the uncoupling activity of SrUCPB was higher than that of SrUCPA in yeast cells (Ito et al., 2006). However, their physiological and biochemical properties in intact mitochondria isolated from the spadix of skunk cabbage have not been examined yet.
In an attempt to clarify the role of plant UCPs in heat generation, SrUCPA and SrUCPB were investigated in thermogenic spadix of skunk cabbage. In this paper, evidence is presented that SrUCPA, not SrUCPB, is the major uncoupling protein in spadix tissues, based on expression profiles and the detailed biochemical characterization of SrUCPA in spadix mitochondria of skunk cabbage. These data are inconsistent with a recent proposal that SrUCPB is a novel thermogenic factor in skunk cabbage (Onda et al., 2008).
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Plant materials
The skunk cabbage (Symplocarpus renifolius) sampled for these experiments was grown in the marshlands of Hakuba village in Nagano Prefecture, Shizukuishi town and Nishiwaga town in Iwate, and Omori town in Akita, Japan. Potato tubers (Solanum tuberosum L. cv. Danshakuimo) were purchased from a local market.
Isolation of mitochondria from skunk cabbage
Florets peeled from 20–30 g of the spadices with a razor blade were ground using a motor-driven blender for 5 s at 4 °C in 60 ml of grinding medium [0.4 M mannitol, 25 mM MOPS–KOH, 2 mM EDTA, 10 mM KH2PO4, 1% PVP-40, 20 mM ascorbic acid, 4 mM cysteine, 2 mM pyruvate, 1% bovine serum albumin (BSA; fatty acid free), 2% PVPP, pH 7.2]. The homogenate was filtered through eight layers of Miracloth (Calbiochem). Cell debris was separated by centrifugation at 1000 g for 10 min at 4 °C and the supernatant was centrifuged again at 10 000 g for 20 min at 4 °C. The organelle pellet was then washed with wash buffer (0.4 M mannitol, 10 mM MOPS–KOH, 2 mM pyruvate, pH 7.2) containing 1% BSA (fatty acid free) by repeating the 1000 and 10 000 g centrifugation steps. The final organelle pellet was resuspended in wash buffer containing 0.1% BSA (fatty acid free), and loaded onto a Percoll step gradient consisting of a 1:3:3 ratio, bottom to top, of 60%:45%:27% Percoll in sucrose wash buffer [250 mM sucrose, 10 mM HEPES–KOH, 2 mM pyruvate, 0.2% BSA (fatty acid free), pH 7.2]. The gradients were centrifuged at 30 000 g for 60 min at 4 °C, and isolated mitochondria appeared as an opaque band at the 27%:45% interface. This band was aspirated and washed by centrifugation at 10 000 g for 10 min at 4 °C and the resulting pellet was then resuspended in wash buffer containing 0.1% BSA (fatty acid free).
Isolation of mitochondria from potato tubers
Mitochondria were isolated from potato tubers using differential centrifugation and Percoll density gradient centrifugation as previously described (Considine et al., 2003).
In vitro expression of SrUCPA and SrUCPB proteins with/without His6-tag
Linear DNA template for in vitro translation of SrUCPA and SrUCPB was obtained by PCR using an RTS wheat germ linear template generation set (Roche Applied Science). In vitro translation of SrUCPA and SrUCPB proteins was performed using the Rapid Translation System RTS100 (Roche Applied Science), according to the manufacturer's instructions for 12 h at 24 °C under vigorous agitation.
Submitochondrial localization of SrUCPA in mitochondria
Mitochondrial proteins (40 µg) were suspended in 20 µl of reaction medium [10 mM HEPES–KOH, 220 mM mannitol, 70 mM sucrose, 0.05 % BSA (fatty acid free), pH 7.2]. For alkaline extraction (pH 
11.5), 20 µl of mitochondrial suspension was mixed with 20 µl of reaction medium containing 0.2 M Na2CO3, and separated by centrifugation (100 000 g for 30 min at 4 °C). For solubilization of proteins, 20 µl of mitochondrial suspension was mixed with 20 µl of reaction medium containing 2% Triton X-100 (TX100) with 1 M NaCl, and again separated by centrifugation (100 000 g for 30 min at 4 °C).
For the preparation of mitoplasts, aliquots of mitochondria (1 mg) were suspended in 100 µl of reaction medium, and then suspended in 900 µl of swelling buffer (10 mM HEPES–KOH, pH 7.2) for 5 min on ice. Another 9 ml of swelling buffer was then added, and after 30 min on ice the mitochondria were ruptured in a Dounce homogenizer (Wlodawer et al., 1966; Werhahn et al., 2001). Shrinking buffer (3.3 ml) containing 10 mM MOPS–KOH, 1.6 M sucrose, 8 mM ATP, 8 mM MgCl2, pH 7.2 was added next and, after 5 min on ice, the mitochondria were ruptured in a Dounce homogenizer (Sottocasa et al., 1967). Mitoplasts were recovered by centrifugation at 4 °C for 20 min at 15 000 g. Mitochondrial or mitoplast protein preparations (40 µg) were suspended in 20 µl of reaction medium, and treated with or without 1% TX100 in the presence or absence of 0.02 mg ml–1 trypsin. After incubation for 30 min on ice, trypsin inhibitor was added at a final concentration of 0.2 mg ml–1.
Preparation of inside-out submitochondrial particles (IO-SMPs)
IO-SMPs were prepared essentially as described previously by Miroux et al. (1992). Briefly, 1 mg of mitochondria was suspended in 1 ml of high-salt medium containing 5 mM MES–KOH, 0.4 M sucrose, 20 mM MgCl2, pH 6.0. After centrifugation at 10 000 g for 10 min at 4 °C, the pellets were resuspended in 3 ml of high-salt medium and submitted to three periods of 10 s of sonication separated by 30 s for cooling. Unbroken mitochondria and larger membrane fragments were removed by centrifugation of 48 400 g for 10 min at 4 °C, and then the supernatants were centrifuged at 105 000 g for 60 min at 4 °C. IO-SMPs were recovered as pellet fractions.
Protease digestion of SrUCPA protein in IO-SMPs
IO-SMPs were suspended in reaction medium and incubated for 30 min on ice with trypsin (0–200 µg of trypsin m–1 of reaction medium) in the presence or absence of 1% TX100. Trypsin activity was quenched by the addition of a mixture of protease inhibitors at final concentrations of 2 mM PMSF, 0.1 mg ml–1 TLCK, 4 µg ml–1 aprotinin, 0.2 mg ml–1 trypsin inhibitor, and 10 mM EDTA. The quenched reactions were incubated for 10 min on ice (Jackson et al., 1998).
Determination of the membrane integrity for IO-SMPs
Cytochrome c (CytC) oxidase activity was measured using a CytC oxidase assay kit (Sigma-Aldrich) according to the manufacturer's instructions. Briefly, 15 µg of mitochondrial proteins were incubated with reduced CytC in a medium consisting of 10 mM TRIS–HCl, 120 mM KCl, pH 7.0, with or without 1 mM n-dodecyl β-D-maltoside (DDM). The oxidation of CytC was monitored at 550 nm. The membrane integrity of the IO-SMP was then calculated using the following equation:
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A(total) and A(IO-SMP) indicate the A550/min of IO-SMP with or without DDM, respectively.
Preparation of antibodies
Polyclonal antibodies against SrUCP, SrAOX, and SrME (malic enzyme) were raised in rabbits using synthetic peptides corresponding to the QVKKFFIKEVPN region, the VAGEEKEGKKAE region, and the NRLHDRNETMYYC region, respectively.
SDS–PAGE and immunoblotting
SDS–PAGE was carried out as described previously (Laemmli, 1970). After electrophoretic separation by SDS–PAGE, proteins were electrotransferred to a PVDF membrane (Millipore) using a semi-dry electrophoretic transfer system (ATTO) and blotting buffer (50 mM TRIS–HCl, 1.44% glycine, 20% methanol). Detection of each protein was performed using anti-SrAOX, SrME, SrUCP, CytC (BD Biosciences), or His (Qiagen) antibodies. The signals were visualized using SuperSignal west femto maximum sensitivity substrate (Pierce).
Membrane potential assay
Membrane potential was measured according to the methods described by Hourton-Cabassa et al. (2002). Briefly, mitochondrial membrane potential changes were monitored at 25 °C with 5 µM safranine O by measuring fluorescence at 569 nm when excited at 495 nm (slit width set at 5 nm) on a fluorometer (RF-5300PC, Shimadzu). The standard medium (2 ml) contained: 0.125 M sucrose, 65 mM KCl, 0.33 mM EGTA, 1 mM MgCl2, 2.5 mM KH2PO4, and 10 mM HEPES–KOH, pH 7.2. Routinely, 2 µM oligomycin, 6 µM carboxyatractiloside, and nigericin (400 ng ml–1 protein) were added to the medium unless otherwise indicated, and 5 mM succinate or 1 mM NADH was used as substrate. To modulate membrane potential, 16 µM linoleic acid, 1 mM purine nucleotide (GTP, GDP, ATP, and ADP), 0.1% BSA, and 0.2 µM valinomycin were added as indicated.
Temperature measurements
The spadix and ambient temperatures were measured with a thermal recorder TR-52 (T & D, Nagano, Japan).
RT-PCR of SrUCPA and SrAOX transcripts
RNA was prepared from spadix, spathe, leaf, and root of skunk cabbage. cDNA was prepared from RNA using Advantage RT-for-PCR Kit (Clontech). RT-PCR (denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s or 1 min) was performed on the indicated PCR cycles, followed by 2% agarose gel electrophoresis. The following 5' and 3' primers were used in RT-PCR: 5' primer (5'-CTGACCTTGTTAAAGTTCGAC-3') and 3' primer (5'-GTAGGCTGAATCTCCCATCA-3') for the amplification of SrUCPs, or 5' primer (5'-CGTTACCCCACCGATGTCTT-3') and 3' primer (5'-GTGCCCCTGGTAATGGATGT-3') for the amplification of SrAOX.
Extraction of total protein from various tissues
Spadix and root tissues were ground in liquid nitrogen using a mortar and pestle. Of the resultant powder, 50 mg were suspended in 0.45 ml extraction buffer (330 mM TRIS–HCl, 7.5% glycerol, 5% SDS, pH 6.8). After 10 min incubation on ice, the supernatant was recovered by centrifugation at 10 000 g for 5 min at 4 °C. The protein extract was clarified by TCA precipitation, and the TCA precipitate was suspended in 160 µl or 20 µl of sample buffer (33 mM TRIS–HCl, 10% glycerol, 1.5% SDS, 1% mercaptoethanol, pH 6.8) containing 0.05% protease inhibitor (Sigma) for spadix and root proteins, respectively, and boiled for 5 min.
Quantification of SrUCPA protein in spadix mitochondria
To construct plasmids encoding His6-tagged SrUCPB under the control of the T7 promoter, the coding region of the SrUCPB gene was amplified by PCR using pZ8-2 (Ito, 1999) as template and the oligonucleotides UCPb-NhF (5'-CGCTTAATTAAACATATGACCGGCGATCACGGCCCG-3') and UCPb-NhR (5'-TTAGTTAGTTACCGGATCCCTTAATTTGGCACCTCTTTGATGA-3') as primers for addition of the Nhis tag, or UCPb-ChF (5'-CTTTAAGAAGGAGATATACCATGGGCGATCACGGCCCGAG-3') and UCPb-ChR (5'-TGATGATGAGAACCCCCCCCATTTGGCACCTCTTT-3') for addition of the Chis tag. The amplified Nhis and Chis fragments were then cloned into the the SmaI and NcoI sites of plasmids pIVEX 1.4WG and pIVEX 1.3WG (Roche Applied Science), respectively, resulting in pI1.4SrUb and pI1.3SrUb.
The SrUCPB-Nhis and -Chis proteins were synthesized using pI1.4SrUb and pI1.3SrUb, respectively, in the RTS 500 system (Roche Applied Science) for 24 h at 24 °C according to the manufacturer's instructions. The synthesized protein fractions (1 ml) were separated into pellet (ppt) and supernatant (sup) fractions by centrifugation at 100 000 g for 30 min. Protein fractions containing SrUCPB-Nhis or -Chis were recovered into ppt fractions and solubilized with 1 ml of the extraction-wash buffer (EW buffer) containing 20 mM MOPS–KOH, 6 M guanidine–HCl, 300 mM NaCl, pH 7.2 for 2 h on ice, and mixed with 0.5 ml TALON resin (BD Biosciences), which had been equilibrated with EW buffer. After gentle agitation for 1 h at 4 °C, protein fractions mixed with TALON resin were applied to a 2 ml disposable gravity column (BD Biosciences). This column was then washed with EW buffer, and eluted with EW buffer supplemented with 250 mM imidazole. Fractions containing His6-tagged SrUCPB protein were dialysed against 20 mM MOPS–KOH (pH 7.2) containing, stepwise, 8 M, 4 M, then 2 M urea, and finally substituted with 10 mM TRIS–HCl (pH 7.2) containing 100 mM NaCl and 0.05% DDM.
By varying the SrUCPB-Chis protein levels, a calibration curve was constructed reflecting the relationship between these levels and the relative intensities calculated using a densitograph.
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Results |
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SrUCPA is a major uncoupling protein in spadix mitochondria of skunk cabbage
Two cDNAs potentially encoding two UCPs, SrUCPA and SrUCPB, were identified in the spadix of the thermogenic skunk cabbage (Ito, 1999). Based on the amino acid sequence deduced from the cDNA sequence, the SrUCPA protein shows typical UCP structure with its six transmembrane regions. However, SrUCPB lacking the fifth transmembrane region of SrUCPA harbours only five transmembrane regions (Fig. 1A). First, their expression in the spadix was analysed by semi-quantitative RT-PCR using a primer pair spanning the transmembrane 5 (TM5) region in SrUCPA (Fig. 1A). As shown in Fig. 1B, a 341 bp fragment derived from SrUCPA was easily amplified from total spadix RNA, while amplification of the SrUCPB-specific 236 bp fragment was barely detectable after 35 PCR cycles. Next, the SrUCPA and SrUCPB proteins were synthesized by in vitro translation and they were analysed by SDS–PAGE and immunoblotting in parallel with total spadix mitochondrial proteins from skunk cabbage. The UCP antibodies, specific to the common C-terminal region of SrUCPA and SrUCPB as shown in Fig. 1A, revealed that in vitro synthesized SrUCPA exhibited the same electrophoretic mobility as the uncoupling protein in skunk cabbage mitochondria (Fig. 1C). Taken together, these results indicated that SrUCPA, not SrUCPB, is a major UCP in spadix mitochondria of skunk cabbage.
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SrUCPA is an integral protein of the inner membrane of spadix mitochondria
Mitochondria isolated from skunk cabbage were subjected to alkaline extraction, a treatment that helps solubilize peripheral membrane proteins, and divided into supernatant (containing matrix and peripheral membrane proteins) and membrane fractions (containing integral membrane proteins) by ultracentrifugation (Fig. 2A). CytC, a peripheral membrane protein from the intermembrane space, and SrME (malic enzyme), a matrix protein, were recovered as expected in the supernatant fraction, while SrUCPA and SrAOX (alternative oxidase), another thermogenic protein in plants, were recovered in the membrane fraction. All of the proteins were solubilized in TX100 treatment. These results indicate that SrUCPA is an integral membrane protein. Then the accessibility of SrUCPA to trypsin hydrolysis was tested (Fig. 2B). Mitochondria and mitoplasts were incubated with 0.02 mg ml–1 trypsin in the presence or absence of 1% TX100. Since mitochondria that have been converted into mitoplasts by osmotic shock release CytC, very little CytC was observed in the mitoplast fraction. In the controls without trypsin, no proteins were degraded regardless of the presence or absence of 1% TX100 (Fig. 2B, lanes 1, 3, 5, and 7). In the presence of both trypsin and TX100, almost all proteins were degraded by trypsin (Fig. 2B, lanes 4 and 8). SrUCPA was not degraded by trypsin in intact mitochondria (Fig. 2B, lane 2) but was degraded in mitoplasts (Fig. 2B, lane 6). Furthermore, as expected, SrAOX and SrME were not degraded in either mitochondria or mitoplasts (Fig. 2B, lanes 2 and 6) because AOX is not exposed to the intermembrane space and ME is localized in the matrix (ap Rees et al., 1983; Affourtit et al., 2002; Albury et al., 2002). Hence, the present results indicate that SrUCPA is an integral membrane protein and is localized in the inner membrane of spadix mitochondria.
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Topological assay of SrUCPA protein structure
Since very little is known about the structural features of plant UCPs, it was considered important to study the structural features of the SrUCPA as a representative of plant UCPs. Accessibility to trypsin digestion of SrUCPA in IO-SMPs was used to probe the protein's topology using the structure of mammalian UCP1 as a model. IO-SMPs were treated with increasing doses of trypsin and SrUCPA was analysed by immunoblotting using the UCP-cab antibody and an antibody against the alternative oxidase, SrAOX (Fig. 3A). The membrane integrity of IO-SMP in each trypsin treatment was confirmed by monitoring CytC oxidase activity. If SrUCPA has a similar topology to UCP1 as shown in Fig. 3B, the trypsin digestion experiments should yield fragments of 25.4 kDa, 14.6 kDa, and 4.9 kDa resulting from digestion at residues R68 in the first matrix oriented loop, K170 in the second one, and K261 in the third one, respectively. As the dose of trypsin was increased, the 33 kDa SrUCPA was degraded, resulting in the successive appearance of two fragments of
26 kDa and 13 kDa. These two fragments could correspond to the 25.4 kDa and 14.6 kDa fragments expected from the model shown in Fig. 3B. However, the existence of a 13 kDa fragment as a final product suggests that the third intramatrix loop in SrUCPA is resistant to the trypsin digestion because of a conformational hindrance in this region or the protection by other molecules. It was confirmed that SrUCPA was completely digested by 200 µg ml–1 trypsin in IO-SMP treated with TX100. Alternative oxidase, as the positive control, which has a large part exposed to the mitochondrial matrix (Affourtit et al., 2002; Albury et al., 2002), was degraded by trypsin treatment in IO-SMPs. The present data support the proposed transmembrane topology of SrUCPA shown in Fig. 3B.
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Determination of the effects of linoleic acid (LA) and purine nucleotides (PN) on the membrane potential of spadix mitochondria using safranine fluorescence
To determine the effects of LA or PN (GTP, GDP, ATP, and ADP), inductors of UCP activity, on the membrane potential mediated by SrUCPA, safranine fluorescence was utilized in the presence of inhibitors of ATP synthase, ADP/ATP carrier, and AOX (oligomycin, atractyloside, and n-propyl gallate, respectively) (Fig. 4A). The mitochondrial membrane potential can be visualized by a decrease in safranine fluorescence upon addition of mitochondria to the medium. Subsequent addition of LA results in a decrease of the membrane potential as expected from increased UCP activity. Addition of PNs, including GTP, GDP, ATP, and ADP, did not change the membrane potential, whereas addition of BSA caused an increase of membrane potential presumably by binding LA and removing it from the mitochondrial membrane (Spector et al., 1969). As a control valinomycin, a membrane potential inhibitor was added and the expected increase of fluorescence, indicative of membrane potential disruption, was observed. To test the experimental set-up, the membrane potential of potato mitochondria was analysed in a similar fashion, except that succinate was used as a respiratory substrate (Fig. 4B). The present results are consistent with observations previously reported by Hourton-Cabassa et al. (2002). Since ATP exerts several effects on mitochondria oxidizing succinate in the presence of succinate, ATP-dependent repolarization observed here is likely to be UCP-independent (Hourton-Cabassa et al., 2002). These results suggest that SrUCPA is involved in LA-induced depolarization, but not in PN-dependent repolarization, in skunk cabbage mitochondria, in a similar way to StUCP in potato mitochondria. It is noteworthy that the response of SrUCPA to the addition of BSA was considerably less sensitive than that of potato UCP (StUCP). This lower sensitivity to BSA was not a result of mitochondrial dysfunction, since a maximal uncoupling rate could still be induced in these preparations by the addition of valinomycin.
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Expression of SrUCPA and SrAOX transcripts in various tissues and thermogenic stages
To explore the transcriptional regulation of SrUCPA under natural growth conditions, RT-PCR was performed using RNA isolated from various tissues (Fig. 5A) or stages (Fig. 5B) of skunk cabbage grown outdoors. The various thermogenic stages considered are listed in Table 1. The spadix during the female stage maintains its temperature constant around 20 °C, despite the wide variation in air temperature, while the spadix temperature during the male stage changes together with the air temperature (Fig. 5B, lower panel). Furthermore, since the difference between the female spadix and ambient temperatures is larger in early morning than during the day (Table 1), thermogenesis in early morning should be higher than that in the day. Therefore, the expression of SrUCPA in female spadix in the early morning and in the daytime was compared with the expression in the male spadix as a control. As shown in Fig. 5C, SrUCPA mRNA, as assessed by semi-quantitative RT-PCR, was constitutively expressed in various tissues and stages regardless of the thermogenic levels. No expression of SrUCPB was detectable with 30 cycles of PCR, in any tissues and stages, even in the spadices during early morning, which is in the highest thermogenic state among the samples examined (Fig. 5C). Since AOX is considered to be another thermogenic factor in plants, gene expression of SrAOX was also examined (McIntosh, 1994). In contrast to SrUCPA, SrAOX was expressed specifically in the spadix (Fig. 5C). In addition, the expression level is higher in the female spadix than in the male spadix, but no difference between early morning and daytime was observed.
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Abundance of SrUCPA and SrAOX proteins in female spadix during thermogenesis
To assess whether SrUCPA and SrAOX proteins show the same expression pattern as their mRNA (Fig. 5C) in various tissues and thermogenic stages, total protein was extracted and analysed by Coomassie Brilliant Blue (CBB) staining (Fig. 6A) and immunoblotting (Fig. 6B) using the UCPcab antibody and an antibody against SrAOX. As shown in Fig. 6A, in CBB staining, the protein band patterns between spadix and root differ markedly from each other, but female and male spadix in the early morning and in the daytime show a similar pattern of protein bands. For undetermined reasons it was not possible to extract total proteins from the leaf and the spathe (data not shown). SrUCPA and SrAOX proteins could be identified in total protein extracts by using the UCPcab and SrAOX antibodies (Fig. 6B). Both proteins were expressed in thermogenic spadix, but not in the root. In addition, the expression level is higher in the female spadix than in the male spadix, but no difference between early morning and daytime was observed. It is worthwhile to note that the observed amount of SrUCPA protein, enriched in the thermogenic tissue during the thermogenic stage, does not parallel that of SrUCPA transcripts which are present in all tissues and stages. However, the observed amount of SrAOX protein followed the transcript abundance in the female spadix. These results indicate that SrUCPA and SrAOX protein are specifically co-expressed in the thermogenic female spadix.
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SrUCPA is highly expressed in spadix mitochondria
While the thermogenic function of UCP1, the uncoupling protein in mammalian BAT, is well established, the physiological role of UCP1 homologues (UCP2 and UCP3 in mammals, plant UCP) in vivo continues to be a matter of intense debate. It is noteworthy that UCP1 itself is present at very high levels at its site of expression and is far more abundant than either UCP2 or UCP3 (Pecqueur et al., 2001; Krauss et al., 2005). Because UCP1, in cold-adapted rat, can represent up to 5% of the mitochondrial proteins in brown adipose tissues (Ricquier et al., 1984; Pecqueur et al., 2001; Harper et al., 2002), it was of interest to determine the expression levels of SrUCPA in spadix mitochondria of skunk cabbage. Quantitative immunoblotting was carried out using purified His6-tagged SrUCPB proteins as a reference. His6-tagged SrUCPB was used because the yield and purity of His6-tagged SrUCPB was much greater than that of SrUCPA (data not shown), and its electrophoretic mobility is similar to the native SrUCPA (Fig. 7A).
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Both N- and C-terminal His6-tagged SrUCPB products (SrUCPB-Nhis or -Chis) were synthesized in vitro and purified using TALON affinity chromatography. These proteins were then analysed by CBB staining on SDS–PAGE and by immunoblotting with either His or UCP-cab antibodies (Fig. 7B). Since 100 ng of both SrUCPB-Nhis and -Chis generated the same signal intensity in immunoblotting, it suggested that the presence of a His-tag had no effect on the antibody sensitivity toward the protein. Quantitative immunoblotting was carried out using the purified SrUCPB-Chis (Fig. 7C). The intensity of the signal for various quantities of SrUCPB-Chis was quantified and plotted on a graph to draw a regression line (data not shown) which was used to estimate the quantities of SrUCPA present in the spadix mitochondrial protein extracts loaded on the gel. On average, based on three replicates, the SrUCPA protein content of mitochondria was about 3% of total mitochondrial proteins. These results show that SrUCPA is an abundant protein in spadix mitochondria, and it is present at levels comparable to that of UCP1 in cold-adapted rats.
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Discussion |
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In this work, it was shown that SrUCPA, not SrUCPB, is the major uncoupling protein in spadix mitochondria of skunk cabbage and that SrUCPA mRNA is predominantly expressed in all tissues examined. Previously, it was shown by another group that defects in the pre-mRNA processing of uncoupling protein genes in rice resulted in multiple abnormal forms of the transcripts (Watanabe and Hirai, 2002). Therefore, possible defects in splicing might account for the appearance of SrUCPB mRNA. In contrast to our conclusion, a recent paper proposed that SrUCPB, not SrUCPA, functions as the major, novel UCP (Onda et al., 2008). These authors concluded that the major UCP in spadix mitochondria is SrUCPB, based on its electrophoretic mobility without appropriate control. However, using in vitro translated SrUCPA and SrUCPB as the control, compelling evidence was provided that the major UCP in skunk cabbage mitochondria is SrUCPA (Fig. 1C). Provided this UCP contains only five transmembrane regions, two degradation fragments of
26 kDa and 13 kDa should not appear because the C-terminal antigenic region which is predicted to localize in the matrix side can be easily digested by the trypsin treatment (Fig. 3). Taken together, both mRNA and protein expression data consistently support our conclusion that SrUCPA is the major UCP in skunk cabbage. The measurements of mitochondrial membrane potential using safranine show that the addition of BSA does not allow full recovery of these membrane potentials in isolated mitochondria for SrUCPA, but does for StUCP. Since BSA binds free fatty acids (FFAs) and removes them from the mitochondrial membrane (Spector et al., 1969), one possibility is that SrUCPA has a higher affinity for FFAs than StUCP. Alternatively, other BSA-binding factors might be present in skunk cabbage mitochondria. It was also observed that SrUCPA shows poor sensitivity to PNs in terms of FFA-activated proton conductance under resting respiration conditions, which is similar to other UCPs except for UCP1. The inhibition of proton conductance by PNs was observed for UCP1 in BAT mitochondria and also several UCP1 homologues (UCP2, UCP3, plant UCP) in reconstituted proteoliposomes (Sluse et al., 2006). However, conflicting results were obtained for isolated mitochondria in the case of UCP1 homologues for which FFA-activated proton conductance was poorly sensitive to PNs under non-phosphorylating conditions (Sluse et al., 2006). It was previously believed that only the ‘superoxide-activated’ state of UCP1 homologues is inhibited by PNs in isolated non-phosphorylating mitochondria (Echtay et al., 2002, 2003; Considine et al., 2003). Recently, significant advances in our understanding of the regulation of the activity of UCP1 homologues have been reported by Sulse et al. (2006), who showed that coenzyme Q, through its redox state, represents a regulator of the inhibition of FFA-activated UCP1 homologues by PNs under phosphorylating respiration conditions (Jarmuszkiewicz et al., 2004, 2005; Navet et al., 2005).
SrUCPA mRNA is found in all of the tissue and its expression pattern is unchanged even if the spadix is exposed to the cold during early morning (Fig. 5C). By contrast, SrAOX is expressed specifically in the thermogenic stages and tissues (Fig. 5C). Since AOX is considered to be another thermogenic factor in plants (Nagy et al., 1972; Watling et al., 2006), AOX may be involved in the thermogenesis of skunk cabbage. However, in fact, AOX genes in non-thermogenic plants are also induced by low temperature, suggesting that the induction of AOX alone is insufficient for thermogenesis (Ito et al., 1997; Watanabe and Hirai, 2002). In contrast to SrUCPA mRNA, the SrUCPA protein exists only in thermogenic spadix and at the female stage, while the observed amount of SrAOX protein followed its transcript abundance in female spadix (Fig. 6B). It is notable that SrUCPA protein only accumulated in the thermogenic female spadix but not in the non-thermogenic male spadix. As far as is known, non-thermogenic plants do not exhibit such a strict regulation of UCP accumulation. An intriguing hypothesis is that SrUCPA and SrAOX act cooperatively to induce the massive heat generation in spadix mitochondria. Further investigation of SrUCPA and SrAOX should provide insight into the molecular basis for thermogenesis of skunk cabbage.
Although the relative abundance of AOX and UCP proteins has not been determined, there is a number of studies that reported the co-existence of both proteins in non-thermogenic plants (Sluse et al., 1998; Sluse and Jarmuszkiewicz, 2000; Considine et al., 2001; Almeida et al., 2002; Clifton et al., 2005; Borecky et al., 2006). This suggests that another factor may have a regulatory function on AOX and UCP activity in skunk cabbage. It is strongly suspected that the redox state of the ubiquinone pool may have a direct impact on the activation state of the AOX and UCP proteins. This determines electron partitioning between the cytochrome and AOX pathways (Dry et al., 1989), and regulates the redox state of AOX via the disulphide bond which modulates AOX activity (Umbach et al., 1994; Vanlerberghe et al., 1999). In addition to that, as mentioned before, coenzyme Q, through its redox state, represents a regulator of UCP under phosphorylating respiration conditions (Jarmuszkiewicz et al., 2004, 2005; Navet et al., 2005). Therefore, post-translational regulation of AOX and UCP mediated by the Q redox state might be important for determining their massive function leading to the thermogenesis in skunk cabbage.
In cold-adapted rats, UCP1 in fact accounts for as much as 5% of the total BAT mitochondrial proteins, and mediates a proton leak leading to thermogenesis in this tissue (Ricquier et al., 1984). By contrast, after lipopolysaccharide stimulation, UCP2 protein levels increase in the lung but reach only 0.02% of the mitochondrial proteins, which then leads to proton leakage without thermogenesis (Pecqueur et al., 2001). Recently, reports have suggested that the widespread presence of UCP1 homologues in eukaryotes implies that these proteins have functions other than thermogenesis (Fernie et al., 2004; Hourton-Cabassa et al., 2004; Vercesi et al., 2006). The main role of plant UCPs in non-thermogenic plants appears to be the regulation of carbon metabolism or the reduction of oxygen species (Brandalise et al., 2003; Considine et al., 2003). Recently, it was also suggested that Arabidopsis UCP1 might be required for the photosynthetic metabolism of this plant (Sweetlove et al., 2006). Since SrUCPA is an abundant protein in spadix mitochondria, there is little doubt about the physiological importance played by SrUCPA. However, it remains unclear whether the thermogenesis of skunk cabbage is attributable to the high abundance of SrUCPA in the thermogenic spadix. Comparative analyses of protein expression levels among other plants would address this question.
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We wish to thank Atsutoshi Obata, Yuki Inukai, and Akiko Miyazaki for their technical support. We are grateful to Takehito Inaba, Takanori Ito, Abidur Rahman, Matsuo Uemura, and Shigeru Saito for their helpful comments. This work was supported by a Grant-in-Aid for the 21st Century COE program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, by a Grant-in-Aid for Scientific Research from Japanese Society for the Promotion of Science, and by the New Energy and Industrial Technology Development Organization (NEDO) under the sponsorship of the Ministry of Economy, Trade and Industry of Japan.
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