Overexpression of UCP1 in tobacco induces mitochondrial biogenesis and amplifies a broad stress response
© Barreto et al.; licensee BioMed Central Ltd. 2014
Received: 26 February 2014
Accepted: 23 May 2014
Published: 28 May 2014
Uncoupling protein one (UCP1) is a mitochondrial inner membrane protein capable of uncoupling the electrochemical gradient from adenosine-5′-triphosphate (ATP) synthesis, dissipating energy as heat. UCP1 plays a central role in nonshivering thermogenesis in the brown adipose tissue (BAT) of hibernating animals and small rodents. A UCP1 ortholog also occurs in plants, and aside from its role in uncoupling respiration from ATP synthesis, thereby wasting energy, it plays a beneficial role in the plant response to several abiotic stresses, possibly by decreasing the production of reactive oxygen species (ROS) and regulating cellular redox homeostasis. However, the molecular mechanisms by which UCP1 is associated with stress tolerance remain unknown.
Here, we report that the overexpression of UCP1 increases mitochondrial biogenesis, increases the uncoupled respiration of isolated mitochondria, and decreases cellular ATP concentration. We observed that the overexpression of UCP1 alters mitochondrial bioenergetics and modulates mitochondrial-nuclear communication, inducing the upregulation of hundreds of nuclear- and mitochondrial-encoded mitochondrial proteins. Electron microscopy analysis showed that these metabolic changes were associated with alterations in mitochondrial number, area and morphology. Surprisingly, UCP1 overexpression also induces the upregulation of hundreds of stress-responsive genes, including some involved in the antioxidant defense system, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione-S-transferase (GST). As a consequence of the increased UCP1 activity and increased expression of oxidative stress-responsive genes, the UCP1-overexpressing plants showed reduced ROS accumulation. These beneficial metabolic effects may be responsible for the better performance of UCP1-overexpressing lines in low pH, high salt, high osmolarity, low temperature, and oxidative stress conditions.
Overexpression of UCP1 in the mitochondrial inner membrane induced increased uncoupling respiration, decreased ROS accumulation under abiotic stresses, and diminished cellular ATP content. These events may have triggered the expression of mitochondrial and stress-responsive genes in a coordinated manner. Because these metabolic alterations did not impair plant growth and development, UCP1 overexpression can potentially be used to create crops better adapted to abiotic stress conditions.
Mitochondrial uncoupling protein one (UCP1) is a nuclear-encoded protein located in the mitochondrial inner membrane. In the presence of fatty acids, UCP1 uncouples the electrochemical gradient from adenosine-5′-triphosphate (ATP) synthesis, dissipating energy as heat . Mammalian UCP1 has long been investigated in brown adipose tissue (BAT) for its role in thermogenesis and the regulation of reactive oxygen species (ROS) production [2–4].
UCP1 has also been found in plants . Similar to its mammalian orthologs, plant UCP1 belongs to a multigenic family whose members are expressed in a time- and tissue-dependent manner and in response to low temperature [6–8]. Plant UCPs have also been shown to be involved in thermogenesis regulation in skunk cabbage  and climacteric increases in respiration in fruits , but the widespread presence of this protein in eukaryotic organisms suggests that it may have other functions . The overexpression of Arabidopsis thaliana UCP1 (AtUCP1) in tobacco plants resulted in increased tolerance to oxidative stress . In addition, the tobacco plants overexpressing AtUCP1 exhibited faster germination under control and stressful conditions, improved performance under drought and salt stresses, and increased rates of photosynthesis . The mechanism underlying this increased stress protection is generally associated with decreased ROS production [11, 12], but a recent study performed on Solanum lycopersicum (tomato) plants overexpressing a UCP gene suggest a wider role for UCPs’ protective mechanisms by altering cell redox homeostasis and antioxidant capacity . These previous studies regarding UCP overexpression, along with a study of an insertional knockout of AtUCP1 , suggest that UCPs may alter metabolism more globally, modulating mitochondrial, chloroplastic and cytosolic metabolism.
The role of mitochondria in energy metabolism and the stress response implies that this organelle communicates with other cellular compartments. Alteration of mitochondrial function modulates the expression of UCP1 and other nuclear-encoded mitochondrial proteins, including alternative oxidase (AOx) and type II NAD(P)H dehydrogenase, through mitochondria-to-nucleus signaling [15, 16]. Mitochondria-to-nucleus communication has been extensively investigated in mammalian models, in which a signaling process coordinates the expression of genes encoded by the mitochondrial and nuclear genomes through mechanisms known as anterograde (nucleus to organelle) and retrograde (organelle to nucleus) signals . The cytosolic concentrations of Ca2+ and ATP, together with the mitochondrial fission/fusion dynamics and ROS production, play central roles in the antero/retrograde signaling pathways and in the regulation of mitochondrial proliferation . Both anterograde and retrograde signaling have been implicated in mitochondrial biogenesis [19, 20], a process that is well documented in mammalian models  but poorly understood in plants . In plants, the upregulation of nuclear-encoded mitochondrial genes have been seen during inflorescence development, especially during flowering [22–24], but the molecular components involved in the regulation of this process remains elusive. Recently, it has been shown that the pentatricopeptide proteins  and the transcription factors bZIP , WRKY , TCP  and NAC  may contribute to the retrograde regulation of mitochondrial/nucleus communication in plants.
In the present work, we investigated the mechanism of UCP1 action in the stress response. We used molecular, cellular and genomic tools to investigate the molecular and cellular events resulting from the overexpression of AtUCP1 in transgenic tobacco plants. We show that AtUCP1 overexpression increases uncoupled respiration, decreases cellular ATP concentration, alters mitochondrial morphology, and triggers retrograde signaling, activating the expression of mitochondrial- and nuclear-encoded mitochondrial proteins. These changes are accompanied by a broad induction of stress-responsive genes that may help the AtUCP1-overexpressing cells reduce the levels of ROS and perform better under stress conditions. The results are discussed in the context of the link between mitochondrial biogenesis and the stress response in plants.
Overexpression of the Arabidopsis UCP1 (AtUCP1) in tobacco shows no phenotypic alterations independent of its transgene expression levels
The transgenic lines P49 and P07 presented 2- to 8-fold increases in AtUCP1 expression compared to WT plants in the two leaves sampled (Figure 1B). Immunoblotting using an anti-AtUCP1 polyclonal antibody, which recognizes the tobacco and Arabidopsis UCP1, showed that the AtUCP1 protein (approximately 32 kD) was weakly detectable in WT leaves but accumulated at high levels in P49 and P07 transgenic lines (Figure 1C).
AtUCP1overexpression triggers mitochondria biogenesis through retrograde signaling
AtUCP1overexpression increases uncoupling respiratory capacity and decreases cellular ATP content
Altered mitochondrial metabolism due to increased UCP1 activity leads to changes in mitochondrial number, volume and morphology
AtUCP1overexpression induces broad transcriptomic activation of mitochondrial and stress-responsive genes
UCP1 transgenic line performed better under abiotic stresses
The mitochondrial respiratory chain is composed of four macromolecular complexes through which free energy is conserved by coupling electron transport to the formation of a proton motive force by complexes I, III, and IV, which is then dissipated by F1FO-ATPase (complex V) for ATP synthesis . In the BAT, electron transport is uncoupled from ATP synthesis by UCP1 for heat production . However, BAT is a specialized tissue dedicated to thermogenesis regulation whose main characteristic is a dramatic increase in mitochondria number and UCP1 expression . In other cell types, however, decreased ATP concentration may compromise cell metabolism ; therefore, cells somehow sense the cytoplasmic ATP concentration as a signal to change gene expression to quickly readjust the mitochondrial architecture and cell metabolism [18, 52]. In this work, we show that the overexpression of UCP1 increases uncoupled respiration and decreases cellular ATP concentration. There is evidence demonstrating that mitochondria isolated from potato plants overexpressing UCP exhibit increased uncoupling respiration , but the present study represents the first time that the effect of UCP on intracellular ATP level has been quantified in plants. It is possible that the enhanced uncoupled respiration and its consequent negative effect on the ATP production trigger a signal to compensate for the energy metabolism and maintain ATP at safe levels. Consistent with this view is the remarkable upregulation of electron transport chain components, including NADH-DeH, NADH-IS2-7, and COX, observed in the P07 overexpressor. The coordinated upregulation of the electron transport chain components encoded by the nuclear and mitochondrial genomes occurs in tissues with high metabolic demands and increased mitochondrial biogenesis [21, 50, 53], such as flowers [22–24, 54] and germinating seeds .
Mitochondrial biogenesis was observed using multiple approaches, including MitoTracker RED fluorescence imaging, quantitation of mtDNA and TEM. In all cases, these methods allowed us to conclude that the overexpression of AtUCP1 resulted in increased mitochondrial number and volume. To assess whether the upregulation of mitochondrial genes was restricted to the electron transport chain components, we performed global transcriptome sequencing and observed the upregulation of hundreds of genes encoding mitochondrial proteins. All of the identified transcripts encoded on the mtDNA were upregulated in transgenic plants, which is consistent with the increased mtDNA content. For instance, the ATP synthase gene encoded by the mtDNA was found to be upregulated by 8.1-fold, and two of its nuclear subunits were increased by 2.7- and 3.6-fold. This upregulation was not restricted to the components involved in the oxidative phosphorylation but also included genes that participate in the tricarboxylic acid cycle (TCA). The key control point of the TCA is the enzyme IDH , which showed increased protein levels in P07 plants (Figure 2G). In fact, experiments performed with mitochondria isolated from potato plants demonstrated that UCP1 activity facilitates higher TCA flux by decreasing membrane potential and, consequently, ROS production .
Transcriptional regulation is the main regulatory factor in the expression of nuclear-encoded genes associated with respiration , but the vast majority of proteins targeted to the mitochondria are transported post-translationally by tightly regulated machinery . Among these proteins, HSP70 is involved in the transport from the cytoplasm to the mitochondrial outer membrane, whereas the mitochondrial TOM and TIM translocases are responsible for transport inside the organelle . Interestingly, all of these proteins were upregulated in the P07 AtUCP1 overexpression line. The increased expression of components of the mitochondrial energy production machinery, along with the increased mitochondrial number of transgenic cells, may alter cellular energy homeostasis. Therefore, the mitochondria may consume more substrates in an attempt to boost ATP synthesis; for that purpose, these substrates must be supplied from other sources. This need might explain the increased photosynthetic rate exhibited by these plants under control conditions . Transcriptome sequencing provides an important tool to investigate this possibility. We did not observe differential expression patterns between WT and P07 plants regarding chloroplast-targeted proteins (data not shown), but further analysis of the transcriptome might provide new insights into this issue.
The adaptation to changes in energy supply and demand influences not only mitochondrial gene expression but also mitochondrial architecture and morphology . Alterations in mitochondrial morphology during starvation have already been observed in Arabidopsis thaliana cells cultured without a carbon source . In human cells, treatment with the chemical uncoupler FCCP causes an increase in oxygen consumption and a decrease in ATP generation, provoking mitochondrial fragmentation and the appearance of donut-shaped mitochondria . This fragmentation may occur to support an increase in respiration in an attempt to elevate ATP levels , whereas the formation of the donut-shaped morphology may be a component of a protective mechanism that helps to preserve the organelles from damage under conditions of metabolic stress [41, 43]. When FCCP is washed out of these cells, the donut morphology persists until the ATP levels are completely restored, at which point the donuts fragment and new mitochondria arise. The mechanisms underlying the donut formation in UCP1-overexpressing cells might be explained by an analogy with human cells. Increased UCP1 activity decreases the membrane potential that is used for ATP synthesis, provoking an increase in mitochondrial number to balance the ATP levels in transgenic plants. Mitochondria are responsible for at least 90% of cell oxygen consumption . We therefore expect that an increase in respiratory chain machinery and mitochondrial number, together with the constant leak caused by UCP1, would result in increased oxygen consumption per molecule of ATP generated. Thus, this metabolic stress caused by UCP1 overexpression appears sufficient to allow the formation of donut mitochondria in plant cells.
Communication between mitochondria and the nucleus is required for the adaptations that we observed in P07 transgenic plants. Although several important regulators of chloroplastic retrograde signaling have been extensively studied , relatively little is known about the regulators of mitochondrial retrograde signaling in plants. Several recently published studies suggest roles for the WRKY, bZIP, TCP, and NAC transcription factor families in activating the expression of nuclear-encoded mitochondrial proteins in plants [26–29]. These transcription factors recognize elements that are overrepresented in the regulatory region of mitochondrial genes and that are involved in several biotic and abiotic stress responses . The colocalization of proteins in mitochondria and the nucleus appears to be an important topic that is very little explored in plants. For example, an Arabidopsis thaliana pentatricopeptide protein (PNM1)  was identified in both mitochondria and nucleus that may act to coordinate the expression between the two genomes. The increased expression of transcription factors that belong to the WRKY and TCP families, along with the upregulation of a large number of pentatricopeptide proteins in transgenic plants, presents the opportunity to further explore new candidates involved in mitochondrial retrograde signaling. The expression of most transcriptional regulators is further linked to cellular signals and environmental cues . The most studied cellular signals involved in mitochondria-nucleus signaling are ATP, calcium and ROS . Although the role of mitochondria in calcium metabolism has been extensively studied in mammals, little is known about calcium signaling by plant mitochondria . Mitochondria in mammals are able to sequester cytoplasmic Ca+2, functioning as a transient calcium store for protective mechanisms [18, 62]. Additionally, the Ca+2 levels inside the organelle are positively correlated with increased ATP production , although Ca+2 overaccumulation is linked to the induction of apoptosis in both mammals and plants . Consistent with this relation, it is very interesting that we found two genes upregulated in transgenic plants that are possible orthologs, an MCU and an MCX, along with several genes involved in calcium signaling (Additional file 3: Tables S2 and Additional file 4: Table S3). The MCUs and MCXs have been extensively studied in animal cells, and their expression is tightly linked with mitochondrial calcium transport [64, 65].
The main focus of previous studies regarding UCP1 overexpression in plants has been ROS production [11–13, 40]. Both potato and tobacco transgenic plants presented decreased superoxide production under abiotic stresses [11, 12, 40]. Additionally, tomato plants overexpressing UCP1 demonstrate not only an alteration on ROS production but also a possible role of UCP1 in regulating the cellular redox homeostasis . The plants analyzed in the present study showed increased expression not only of the antioxidant defense system, including GPX, GSTs and SOD, but also of an array of stress-responsive genes. This effect may help explain the positive impact of AtUCP1 overexpression on the tolerance to multiple abiotic stresses. Consistent with previous data, we show in this work that AtUCP1 overexpression protects plants from a number of abiotic stresses, including high pH, low pH and cold. The fact that transgenic tobacco performs better at low temperatures when the medium is supplemented with fatty acids reinforces the thermogenic role of UCP1 in plants. The importance of ROS in cell signaling is well known ; thus, the results regarding decreased mitochondrial ROS production under oxidative, osmotic and salt stresses also reinforce the importance of this molecule in UCP1-mediated retrograde signaling.
Together, these data indicate that AtUCP1-overexpressing lines suffer metabolic stress caused by the increased uncoupling respiration. Chronic overexpression of UCP1 can induce mitochondrial biogenesis in mammals . Therefore, we propose that in the transgenic plants, the lower ATP level acts as a key element in the retrograde signaling, promoting a broad increase in the expression of both mitochondrial genes and stress-related genes. This pattern is reflected in the decreased ROS production and the better performance of these plants under various stresses. These findings advance our understanding of stress-tolerance mechanisms, mitochondrial biogenesis and bioenergetic adaptation in plants and therefore might assist in the implementation of biotechnological tools for the development of abiotic stress-tolerant plants. The overall regulation of stress-responsive genes indicates a link between UCP1-activated mitochondrial biogenesis and the increased stress response exhibited by the transgenic plants.
In this work, we present strong evidence that the overexpression of UCP1 protein affects the mitochondrial dynamics at both structural and metabolic levels, leading to an increased mitochondria number and volume per cell. It could be argued that these changes are a consequence of the overpopulation of UCP1 protein at the inner mitochondrial membrane, thereby leading to abnormal mitochondrial structure and function. We interpret the results presented in this manuscript as a beneficial effect of the overexpression of UCP1 that triggers a retrograde signaling process, signaling the nuclear and mitochondrial genomes to increase the production of mitochondrial proteins. Consequently, more mitochondrial membrane is produced. The increase in UCP1, both because of the overproduction of the protein and the increased mitochondrial number and volume, leads to increased uncoupled respiration, decreasing the cellular ATP concentration. This change may trigger metabolic stress and enhances a strong stress response. Because it does not affect plant growth and development, this mechanism might be used to create crops better adapted to abiotic stress conditions.
Plant materials and growth conditions
Tobacco (Nicotiana tabacum) ecotype SR1 overexpressing AtUCP1 was obtained as previously described . In this work, we used two independent transgenic lines, P49 and P07, which express AtUCP1 at intermediate and high levels, respectively. Transgenic and wild-type (WT) plants were grown under a light/dark cycle of 16 h at 100 μE m−2 s−1 and 8 h in darkness for 12 weeks on 50:50 soil/vermiculite mixtures at 24°C. The plants were fertilized weekly with half-strength MS medium not supplemented with sucrose. For the cellular and molecular analysis, samples of the five youngest leaves (L1 to L5) of each genotype were collected, frozen in liquid nitrogen, and stored at −80°C. For the other experiments, seeds were grown directly in experimental medium, as described in the figure legends.
For mitochondrial DNA quantification, total DNA was isolated from WT, P49, and P07 plants using the plant DNAzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For the mRNA expression analysis, total RNA was isolated from leaf tissue using the NucleoSpin Mini RNA/Protein Kit (Macherey-Nagel, Duren, Germany). cDNA was synthesized using the Revertaid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) according to the manufacturer’s protocol. qRT-PCR was performed using an ABI PRISM 7500 (Applied Biosystems, Foster City, CA, USA) with SYBR Green (Applied Biosystems). The reactions were performed at least in triplicate for four biological replicates. The mRNA expression and mtDNA content analysis were normalized using ACTIN1 as an internal reference. Values are presented as the mtDNA content or transcript abundance of P49 and P07 relative to WT. Student’s t-test was performed to determine significance (p < 0.05). Primers based on the MATR gene were used for mitochondrial DNA quantification .
The following primers were designed for amplification:
Total soluble proteins were extracted from WT, P49 and P07 plants by grinding frozen leaf tissue in 5× protein sample buffer (40 mM Tris, pH 7.4; 30 mM NaCl; 10 mM β-mercaptoethanol; 0.1% Triton X-100; 5 mM benzamidine). The proteins were separated on 12% SDS-PAGE gels, transferred to nylon membranes, and blotted using the following polyclonal antibodies raised against Arabidopsis proteins (Agrisera, Vännäs, Sweden): COXII, VDAC (At3g01280), AOx1/2 (At2g22370 and At5g64210), IDH (At4g35260 and At2g27130), and ACTIN (At2g37620, At3g18780, At3g53750, At5g59370, At2g42100, At5g09810, At1g49240, At3g12110, and At3G46520). The bands were detected using an ImageQuant LAS500 (GE Healthcare, Little Chalfont, UK) with the SuperSignal West Pico chemiluminescent substrate (Thermo-Scientific, Waltham, MA, USA).
Mitochondrial quantification in leaf mesophyll protoplasts by MitoTracker Red staining
Tobacco mesophyll protoplasts were isolated from WT, P49, and P07 plants as previously described , with minor modifications. Leaf strips (0.1 cm in length) were vacuum-infiltrated and incubated for 3 h in an enzyme solution (20 mM MOPS, pH 5.7; 0.4 M d-mannitol; 20 mM KCl; 10 mM CaCl2; 0.1% BSA; 1.5% cellulase; 0.4% macerozyme). After digestion, the suspension was filtered through 75 μm nylon mesh and centrifuged at 100 × g for 2 min. The protoplasts were resuspended in W5 buffer (2 mM MOPS, pH 5.7; 154 mM NaCl; 125 mM CaCl2; 5 mM KCl), incubated on ice for 30 min, and subsequently resuspended in MMG solution (4 mM MOPS, pH 5.7; 0.4 M mannitol; 15 mM MgCl2). The protoplasts were then stained with 250 nM MitoTracker Red CM-H2XRos (Invitrogen) for 30 min in the dark and washed in MMG solution before viewing. MitoTracker Red CM-H2XRos fluorescence was detected at Ex/Em of 543/589 nm and chlorophyll auto fluorescence was detected at Ex/Em 543/645 nm using a confocal microscope (LSM780-NLO; Zeiss, Oberkochen, Germany) equipped with a 40× oil-immersion objective. The image analysis was performed for 40 protoplasts per sample using ImageJ 1.44p . MitoTracker Red CM-H2XRos fluorescence intensity was normalized using the chlorophyll autofluorescence intensity.
Mitochondrial superoxide detection by MitoSox staining
Leaf discs (0.8 cm in diameter) isolated from L3 leaves were treated with half-strength MS medium (control) or half-strength MS medium supplemented with 100 mM NaCl (salt stress) or 10 mM H2O2 (oxidative stress) for 20 h under constant light and agitation. For root analysis, plants grown for 3 weeks on half-strength MS medium were transferred to petri dishes containing fresh half-strength MS medium not supplemented (control) or supplemented with 100 mM NaCl (salt stress) or 100 mM mannitol (hyperosmotic stress) and kept under constant light for 20 h. The leaf discs and roots were stained with 5 μM MitoSox-Red (Invitrogen) for 25 min and washed with half-strength MS medium before viewing. Fluorescence was detected at Ex/Em of 510/580 nm using a confocal microscope (LSM780-NLO; Zeiss) equipped with a 10× objective.
Transmission electron microscopy
Leaf strips (1–2 mm in length) from L2 leaves of WT and P07 plants were submerged and vacuum infiltrated for at least 2 h in fixative solution (2.5% glutaraldehyde, 2.5% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4). The samples were washed in 0.1 M cacodylate buffer and postfixed in a solution containing 1% osmium tetroxide (OsO4) and 1.5% potassium ferrocyanide (KFeCN6) for 1 h. The samples were then washed 3 times in water and incubated in 1% uranyl acetate in maleate buffer for 1 h; this was followed by 3 washes in maleate buffer and subsequent dehydration in a graded ethanol series (50%, 70%, and 90%, 10 min each; 100%, 2 × 10 min). The samples were then placed in propylene oxide for 1 h and infiltrated in a 1:1 mixture of propylene oxide and TAAB 812 Resin mixture (Marivac Canada, St. Laurent, Canada). The samples were embedded in TAAB 812 Resin mixture and polymerized at 60°C for 48 h. Ultrathin sections of approximately 90 nm were cut using a Reichert Ultracut-S microtome, placed onto copper grids, stained with lead citrate, and examined using a Tecnai G2 Spirit BioTWIN transmission electron microscope. The images were recorded with an AMT 2 k CCD camera. The mitochondrial number and area were quantified on 20 field images representing each genotype at 4800× direct magnification. The image analysis was performed using ImageJ 1.44p .
ATP assays were performed on L4 and L5 leaves isolated from four biological replicates of WT and P07 plants. ATP extraction was performed as previously described , with minor modifications. The leaves were ground in liquid nitrogen, and 30 mg of leaf tissue was homogenized with 300 μL of 0.1 M HCl for 5 min. The homogenate was centrifuged at 20,000 × g for 10 min, and the supernatant was centrifuged using a microconcentrator Ultra-15 (Millipore, Billerica-USA) at 14,000 × g for 20 min. The ATP content was determined by using an ATP assay kit (Invitrogen) and a GloMax luminometer (Promega, Fitchburg, WI, USA).
Measurement of mitochondrial respiration
Measurements of mitochondrial respiration were performed using crude isolated mitochondria from six biological replicates of WT and P07 plants. The isolation of crude mitochondria was performed as previously described , with minor modifications. All steps were performed at 4°C. Approximately 5 g of leaves were cut with a razor blade and ground in 20 mL of grinding buffer (0.3 M sucrose, 60 mM TES, 10 mM EDTA, 10 mM KH2PO4, 25 mM disodium pyrophosphate, 1 mM glycine, 1% (w/v) PVP-40, 1% (w/v) BSA, 50 mM sodium ascorbate, 20 mM cysteine, pH 8.0). The extract was filtered through two layers of 20 μm nylon mesh and one layer of Miracloth (Millipore, Billerica, MA, USA) and centrifuged twice at 2500 × g for 5 min to remove the debris. The resulting supernatant was centrifuged at 15,000 × g for 15 min, and the resulting pellet was gently resuspended in 20 mL of wash buffer (0.3 M sucrose, 10 mM TES, 2 mM EDTA, 10 mM KH2PO4, pH 7.5) and centrifuged at 15,000 × g for 15 min. The pellet containing the crude mitochondria was resuspended in 200 μL of wash buffer. The respiratory measurements were performed using an Oxygraph-2 k respirometer (Oroboros, Innsbruck, Austria) at 25°C in 2 mL of mitochondria-containing assay buffer (0.3 M sucrose, 10 mM TES, 10 mM KCl, 2 mM MgSO4, 5 mM KH2PO4, 0.1% (w/v) BSA, 2 μM EGTA, pH 7.5). Malate (5 mM) and glutamate (10 mM) were used as substrates to stimulate respiration. ADP (800 μM) was added to measure ADP-dependent respiration. Oligomycin (5 μM) was used as an F1FO ATP synthase inhibitor to measure oxygen consumption due to uncoupling activity, and the chemical ionophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (2 μM) was used to determine the maximum oxygen consumption.
Transcriptome sequencing, assembly, and mapping
Total RNA isolated from four biological replicates of WT and P07 L3 leaves was used to create single-end RNASeq libraries using the Illumina TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The libraries were sequenced in four lanes of an Illumina HiSeq 2000 for 75 cycles. A total of 42.7 Gb of sequence was generated for the 8 libraries, with a minimum of 27.9 million and a maximum of 113.2 million reads for each library (Additional file 6: Table S5). The Illumina reads were filtered to remove adapters and low-quality reads (reads <70% bases with quality ≥ Q20) using AdapterRemoval, the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit), and Perl scripts. After the filtering step, the reads were subjected to digital normalization using the diginorm software (https://github.com/ged-lab/2012-paper-diginorm). The resulting 49.5 million reads were assembled with the Trinity  assembler, generating 271,750 transcript contigs. High-quality reads (569 million reads) were mapped to Trinity contigs using Bowtie , and the RPKM  values were calculated. Because the N. tabacum genome is not yet completely sequenced and the scaffolds of N. benthamiana are estimated to cover only 79% of its genome, we used the complete genome sequence of Solanum lycopersicum, a close relative of N. tabacum, as a template. To identify protein-coding genes, all 271,750 contigs were used as queries in BLASTn and BLASTx searches against both the non-redundant set of 34,727 tomato-coding sequences (CDSs) and predicted protein sequences. The 271,750 contigs were compared to the Solgenomics tomato genome predicted protein database using BLASTx (E-value <1e−5). Of the total contigs, 134,752 mapped to the tomato genome, resulting in the identification of 20,045 distinct orthologs for the tomato CDS models. As representative contigs, we chose the three best matching contigs with higher average RPKM values (P07 and WT) for each tomato CDS. An F-test was used to calculate whether the samples had equal or unequal variances, and the appropriate Student t-test was then applied to calculate the significant difference between the average RPKM of P07 versus WT in a contig-wise manner. The contigs identified as differentially expressed were selected for the next steps. The fold change value for the representative transcript was calculated as the ratio between the average RPKM values in P07 and WT. The subcellular annotation of the defined contigs was determined by BLASTx queries against the TAIR  protein database (release 10) and TargetP . Gene function data were obtained from the existing annotations of the tomato genome. The tomato protein dataset was annotated against UniProt  and mapped against the COG (Eukaryotic Clusters of Orthologous Groups)  database with BLASTp, and COG functional categories were assigned only if the two best hits had the same COG function. Stress-related genes were mapped using BLASTx against the tomato proteome in the Stress Responsive Transcription Factor Database (STIFDB) , and annotations were assigned to the corresponding representative tobacco contig.
Sequence data from this article can be found in the GenBank/EMBL database under the following accession numbers: AtUCP1 (NM_115271.4), Actin1 (EU938079.1), NADH-DeH (Y09109.1), NADH-IS2 (M77225.1), NADH-IS7 (L16810.1), GRPe chaperone (AF098636.1), AtMCU (NP_119075), MmMCU (XP_006513531), HsMCU (NP_612366), AtCCX3 (NP_566474).
Transcriptome sequencing data are available from Sequence Read Archive (SRA), which is accessible through NCBI BioProject ID: PRJNA211804 under the experiment ID SUB287723.
We are grateful to Ana Paula Arruda for her valuable help with transmission electron microscopy, Anibal Vercesi and Paolo la Guardia for their assistance with the respiration assays, and Eduardo Kyota for his help with the immunoblotting experiments. We also thank the staff of the National Institute of Photonics Applied to Cell Biology (INFABIC) of the State University of Campinas (UNICAMP) for their assistance with the confocal microscopy analysis and the staff of the Life Sciences Core Facility (LaCTAD) of the State University of Campinas (UNICAMP) for their assistance with the genomic and bioinformatic analyses. Pedro Barreto was supported by an FAPESP PhD fellowship (12/00126-1). Izabella Neshich was supported by an FAPESP PhD fellowship (12/00235-5). Vagner Okura is a bioinformatician at the Life Sciences Core Facility (LaCTAD) of the State University of Campinas (UNICAMP). Paulo Arruda and Ivan G. Maia are CNPq productivity research fellows.
- Nedergaard J, Ricquier D, Kozak LP: Uncoupling proteins: current status and therapeutic prospects. EMBO Rep. 2005, 6: 917-921. 10.1038/sj.embor.7400532.PubMed CentralView ArticlePubMedGoogle Scholar
- Krauss S, Zhang CY, Lowell BB: The mitochondrial uncoupling-protein homologues. Nat Rev Mol Cell Biol. 2005, 6: 248-261. 10.1038/nrm1592.View ArticlePubMedGoogle Scholar
- Andrews ZB, Diano S, Horvath TL: Mitochondrial uncoupling proteins in the CNS: in support of function and survival. Nat Rev Neurosci. 2005, 6: 829-840. 10.1038/nrn1767.View ArticlePubMedGoogle Scholar
- Kajimura S, Seale P, Spiegelman BM: Transcriptional control of brown fat development. Cell Metab. 2010, 11: 257-262. 10.1016/j.cmet.2010.03.005.PubMed CentralView ArticlePubMedGoogle Scholar
- Vercesi AE, Martins LS, Silva MAP, Leite HMF, Cuccovia IM, Chaimovich H: PUMPing plants. Nature. 1995, 375: 24-10.1038/375024a0.View ArticleGoogle Scholar
- Maia IG, Benedetti CE, Leite A, Turcinelli SR, Vercesi AE, Arruda P: AtPUMP: an Arabidopsis gene encoding a plant uncoupling mitochondrial protein. FEBS Lett. 1998, 429: 403-406. 10.1016/S0014-5793(98)00634-6.View ArticlePubMedGoogle Scholar
- Borecky J, Nogueira FTS, Oliveira KAP, Maia IG, Vercesi AE, Arruda P: The plant energy-dissipating mitochondrial systems: depicting the genomic structure and the expression profiles of the gene families of uncoupling protein and alternative oxidase in monocots and dicots. J Exp Bot. 2006, 57: 849-864. 10.1093/jxb/erj070.View ArticlePubMedGoogle Scholar
- Vercesi AE, Borecký J, Maia IG, Arruda P, Cuccovia IM, Chaimovich H: Plant uncoupling mitochondrial proteins. Annu Rev Plant Biol. 2006, 57: 383-404. 10.1146/annurev.arplant.57.032905.105335.View ArticlePubMedGoogle Scholar
- Ito-Inaba Y, Hida Y, Mora H, Inaba T: Molecular identity of uncoupling proteins in thermogenic skunk cabbage. Plant Cell Physiol. 2008, 49: 1911-1916. 10.1093/pcp/pcn161.View ArticlePubMedGoogle Scholar
- Jezek P, Borecký J, Zácková M, Costa AD, Arruda P: Possible basic and specific functions of plant mitochondrial uncoupling protein (pUCP). Biosci Rep. 2001, 21: 237-245. 10.1023/A:1013660611154.View ArticlePubMedGoogle Scholar
- Brandalise M, Maia IG, Borecký J, Vercesi AE, Arruda P: Overexpression of plant mitochondrial uncoupling protein in transgenic tobacco increases tolerance to oxidative stress. J Bioenerg Biomembr. 2003, 35: 205-209.View ArticleGoogle Scholar
- Begcy K, Mariano ED, Mattiello L, Nunes AV, Mazzafera P, Maia IG, Menossi M: An Arabidopsis mitochondrial uncoupling protein confers tolerance to draught and salt stress in transgenic tobacco plants. Plos One. 2011, 6: e23776-10.1371/journal.pone.0023776.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen S, Liu A, Zhang S, Li C, Chang R, Liu D, Ahammed GJ, Lin X: Overexpression of mitochondrial uncoupling protein conferred resistance to heat stress an Botrytis cinerea infection in tomato. Plant Physiol Biochem. 2013, 73: 245-253.View ArticlePubMedGoogle Scholar
- Sweetlove LJ, Lytovchenko A, Morgan M, Nunes-Nesi A, Taylor NL, Baxter CJ, Eickmeier A, Fernie AR: Mitochondrial uncoupling protein is required for efficient photosynthesis. Proc Natl Acad Sci U S A. 2006, 103: 19587-19592. 10.1073/pnas.0607751103.PubMed CentralView ArticlePubMedGoogle Scholar
- Rhoads DM, Umbach AL, Subbaiah CC, Siedow JN: Mitochondrial reactive oxygen species: contribution to oxidative stress and interorganellar signaling. Plant Physiol. 2006, 141: 357-366. 10.1104/pp.106.079129.PubMed CentralView ArticlePubMedGoogle Scholar
- Rhoads DM, Subbaiah CC: Mitochondrial retrograde regulation in plants. Mitochondrion. 2007, 7: 177-194. 10.1016/j.mito.2007.01.002.View ArticlePubMedGoogle Scholar
- Woodson JD, Chory J: Coordination of gene expression between organellar and nuclear genomes. Nat Rev Genet. 2008, 9: 383-395. 10.1038/nrg2348.View ArticlePubMedGoogle Scholar
- Ryan MT, Hoogenrad MJ: Mitochondrial-nuclear communications. Annu Rev Biochem. 2007, 76: 701-722. 10.1146/annurev.biochem.76.052305.091720.View ArticlePubMedGoogle Scholar
- Pesaresi P, Schneider A, Kleine T, Leister D: Interorganellar communication. Curr Opin Plant Biol. 2007, 10: 600-606. 10.1016/j.pbi.2007.07.007.View ArticlePubMedGoogle Scholar
- Jung HS, Chory J: Signaling between chloroplasts and the nucleus: can a systems biology approach bring clarity to a complex and highly regulated pathway. Plant Physiol. 2009, 152: 453-459.View ArticlePubMedGoogle Scholar
- Welchen E, Garcia L, Mansilla N, Gonzalez DH: Coordination of plant mitochondrial biogenesis: keeping pace with cellular requirements. Front Plant Sci. 2013, 4: 551-PubMed CentralGoogle Scholar
- Zabaleta E, Heiser V, Grohmann L, Brennicke A: Promoters of nuclear-encoded respiratory chain complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen-specific expression. Plant J. 1998, 156: 49-59.View ArticleGoogle Scholar
- Binder S, Brennicke A: Gene expression in plant mitochondria: transcriptional and post-transcriptional control. Philos Trans R Soc Lond B Biol Sci. 2003, 3586: 181-188.View ArticleGoogle Scholar
- Giegè P, Sweetlove LJ, Congat V, Leaver CJ: Coordination of nuclear and mitochondrial genome expression during mitochondrial biogenesis in Arabidopsis. Plant Cell. 2005, 17: 1497-1512. 10.1105/tpc.104.030254.PubMed CentralView ArticlePubMedGoogle Scholar
- Hammani K, Gobert A, Hleibieh K, Choulier L, Small L, Giegè P: An Arabidopsis dual-localized pentatricopeptide repeat protein interacts with nuclear proteins involved in gene expression regulation. Plant Cell. 2011, 23: 730-740. 10.1105/tpc.110.081638.PubMed CentralView ArticlePubMedGoogle Scholar
- Roschzttardtz H, Fuentes I, Vásquez M, Corvalán C, León G, Gómez I, Araya A, Holuique L, Vicente-Carbajosa J, Jordana X: A nuclear gene encoding the iron-sulfur subunit of mitochondrial complex II is regulated by B3 domain transcription factors during seed development in Arabidopsis thaliana. Plant Physiol. 2009, 150: 84-95. 10.1104/pp.109.136531.PubMed CentralView ArticlePubMedGoogle Scholar
- Van Aken O, Zhang B, Law S, Narsai R, Whelan J: AtWKRY40 and AtWRKY63 modulate the expression of stress-responsive nuclear genes encoding mitochondrial and chloroplast proteins. Plant Physiol. 2013, 162: 254-271. 10.1104/pp.113.215996.PubMed CentralView ArticlePubMedGoogle Scholar
- Giraud E, Ng S, Carrie C, Duncan O, Low J, Lee CP, Van Aken O, Millar AH, Murcha M, Whelan J: TCP transcription factors link the regulation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana. Plant Cell. 2010, 22: 3921-3934. 10.1105/tpc.110.074518.PubMed CentralView ArticlePubMedGoogle Scholar
- De Clercq I, Vermeirssen V, Van Aken O, Vandepoele K, Murcha MW, Law SR, Inzé A, Ng S, Ivanova A, Rombaut D, Van de Cotte B, Jaspers P, Van de Peer Y, Kangasjärvi J, Whelan J, Van Breusegem F: The membrane-bound NAC transcription factor ANAC013 functions in mitochondrial retrograde regulation of the oxidative stress response in Arabidopsis. Plant Cell. 2013, 25: 3472-3490. 10.1105/tpc.113.117168.PubMed CentralView ArticlePubMedGoogle Scholar
- Ölçer H, Lloyd JC, Raines CA: Photosynthetic capacity is differentially affected by reductions in sedoheptulose-1,7-biphosphatase activity during leaf development in transgenic tobacco plants. Plant Physiol. 2001, 125: 982-989. 10.1104/pp.125.2.982.PubMed CentralView ArticlePubMedGoogle Scholar
- Rogalski M, Schöttler MA, Thiele W, Schulze WX, Bock R: Rpl33, a nonessential plastid-encoded ribosomal protein in tobacco, is required under cold stress conditions. Plant Cell. 2008, 20: 2221-2237. 10.1105/tpc.108.060392.PubMed CentralView ArticlePubMedGoogle Scholar
- Unseld M, Marienfeld JR, Brandt P, Brennicke A: The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotide. Nat Genet. 1997, 15: 57-61. 10.1038/ng0197-57.View ArticlePubMedGoogle Scholar
- Dickinson A, Yeung KY, Donoghue J, Baker MJ, Kelly RDW, McKenzie M, Jhons TJ, St John JC: The regulation of mitochondrial DNA copy number in glioblastoma cells. Cell Death Differ. 2013, 20: 1644-1653. 10.1038/cdd.2013.115.PubMed CentralView ArticlePubMedGoogle Scholar
- Klodmann J, Senkler M, Rode C, Braun HP: Defining the protein complex proteome of plant mitochondria. Plant Physiol. 2011, 157: 587-598. 10.1104/pp.111.182352.PubMed CentralView ArticlePubMedGoogle Scholar
- Padimam M, Reddy VS, Beachy RN, Fauquet CM: Molecular characterization of a plant mitochondrial chaperone GrpE. Plant Mol Biol. 1999, 39: 871-881. 10.1023/A:1006143305907.View ArticleGoogle Scholar
- Fiorani F, Umbach AL, Siedow JN: The alternative oxidase of plant mitochondria is involved in the acclimation of shoot growth at low temperature. A study of Arabidopsis AOX1a transgenic plants. Plant Physiol. 2005, 139: 1795-1805. 10.1104/pp.105.070789.PubMed CentralView ArticlePubMedGoogle Scholar
- Smolková K, Jezek P: The role of mitochondrial NADPH-dependent isocitrate dehydrogenase in cancer cells. Int J Cell Biol. 2012, 212: 1-12.View ArticleGoogle Scholar
- Robert N, D’Erfurth I, Marmagne A, Erhardt M, Allot M, Boivin K, Gissot L, Monachello D, Michaud M, Duchêne AM, Barbier-Brygoo H, Maréchal-Drouard L, Ephritikhine G, Filleur S: Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria. Plant Mol Biol. 2012, 78: 431-446. 10.1007/s11103-012-9874-5.View ArticlePubMedGoogle Scholar
- Leadsham JE, Sanders G, Giannaki S, Bastow EL, Hutton R, Naeimi WR, Breitenbach M, Gourlay CW: Loss of cytochrome c oxidase promotes RAS-dependent ROS production from the ER resident NADPH oxidase, Yno1p, in yeast. Cell Metab. 2013, 18: 279-286. 10.1016/j.cmet.2013.07.005.View ArticlePubMedGoogle Scholar
- Smith AMO, Ratcliffe G, Sweetlove LJ: Activation and function of mitochondrial uncoupling protein in plants. J Biol Chem. 2004, 279: 51944-51952. 10.1074/jbc.M408920200.View ArticlePubMedGoogle Scholar
- Liesa M, Shirihai OS: Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure. Cell Metab. 2013, 17: 491-506. 10.1016/j.cmet.2013.03.002.View ArticlePubMedGoogle Scholar
- Schneider CA, Rasband WS, Eliceir KW: NIH image to ImageJ: 25 years of image analysis. Nat Methods. 2012, 9: 671-675. 10.1038/nmeth.2089.View ArticlePubMedGoogle Scholar
- Liu X, Hajnóczky G: Altered fusion dynamics underlie unique morphological changes in mitochondria during hypoxia-reoxygenation stress. Cell Death Differ. 2011, 18: 1561-1572. 10.1038/cdd.2011.13.PubMed CentralView ArticlePubMedGoogle Scholar
- The UniProt Consortium: Update on activities at the universal protein resource (UniProt) in 2013. Nucleic Acids Res. 2013, 41: D43-D47.PubMed CentralView ArticleGoogle Scholar
- Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikoslkaya AN, Rao BS, Smirnov AS, Sverdlove AV, Vasudevan S, Wolf YI, Yin JJ, Natale DA: The COG database: an updated version includes eukaryotes. BMC Bioinforma. 2003, 4: 41-10.1186/1471-2105-4-41.View ArticleGoogle Scholar
- Lamesch P, Berardini TZ, Li D, Swarbreck D, Wilks C, Sasidharan R, Muller R, Dreher K, Alexander DL, Garcia-Hernandez M, Karthikeyan AS, Lee CH, Nelson WD, Ploetz L, Singh S, Wensel A, Huala E: The Arabidopsis information resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 2012, 40: 1202-1210. 10.1093/nar/gkr1090.View ArticleGoogle Scholar
- Emanuelsson O, Brunak S, Heijne VJ, Nielsen H: Locating proteins in the cell using TargetP, SignalP, and related tools. Nat Protoc. 2007, 2: 953-971. 10.1038/nprot.2007.131.View ArticlePubMedGoogle Scholar
- Oelkrug R, Kutschke M, Meyer CW, Heldmaier G, Jastroch M: Uncoupling protein 1 decreases superoxide production in brown adipose tissue mitochondria. J Biol Chem. 2010, 285: 21961-21968. 10.1074/jbc.M110.122861.PubMed CentralView ArticlePubMedGoogle Scholar
- Vafai SB, Mootha VK: Mitochondrial disorders as windows into an ancient organelle. Nature. 2012, 491: 374-383. 10.1038/nature11707.View ArticlePubMedGoogle Scholar
- Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S, Lowell B, Scarpulla RC, Spiegelman BM: Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell. 1999, 98: 115-124. 10.1016/S0092-8674(00)80611-X.View ArticlePubMedGoogle Scholar
- Miyoshi N, Oubrahim H, Chock PB, Stadtman ER: Age-dependent cell death and the role of ATP in hydrogen-peroxide-induced apoptosis and necrosis. Proc Natl Acad Sci U S A. 2006, 103: 1727-1731. 10.1073/pnas.0510346103.PubMed CentralView ArticlePubMedGoogle Scholar
- Moghadam AA, Ebrahimie E, Taghavi SM, Niazi A, Babgohari MZ, Deihimi T, Djavaheri M, Ramezani A: How the nucleus and mitochondria communicate in energy production during stress: nuclear MtATP6, an early-stress responsive gene, regulates the mitochondrial F1F0-ATP synthase complex. Mol Biotechnol. 2013, 54: 756-769. 10.1007/s12033-012-9624-6.View ArticlePubMedGoogle Scholar
- Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, Boss O, Michael LF, Puigserver P, Isotani E, Olson EN, Lowell BB, Bassel-Duby R, Spiegelman BM: Transcriptional co-activator PGC-1 alpha drives the formation of slow twitch muscle fibres. Nature. 2002, 418: 797-801. 10.1038/nature00904.View ArticlePubMedGoogle Scholar
- Peters K, Niessen M, Peterhansel C, Spath B, Holzle A, Binder S, Marchfelder A, Braun HP: Complex I-complex II ratio strongly differs in various organs of Arabidopsis thaliana. Plant Mol Biol. 2012, 79: 273-284. 10.1007/s11103-012-9911-4.View ArticlePubMedGoogle Scholar
- Law SR, Narsai R, Taylor NL, Dellanoy E, Carrie C, Giraud E, Millar AH, Small I, Whelan J: Nucleotide and RNA metabolism prime translational initiation in the earliest events of mitochondrial biogenesis during Arabidopsis germination. Plant Physiol. 2012, 158: 1610-1627. 10.1104/pp.111.192351.PubMed CentralView ArticlePubMedGoogle Scholar
- Berg JM, Tymoczko JL, Stryer L: Biochemistry. The Citric Acid Cycle. 2002, New York: W H Freeman, 5Google Scholar
- Pfanner N, Geissler A: Versatility of the mitochondrial protein import machinery. Nat Rev Mol Cell Biol. 2001, 2: 339-349. 10.1038/35073006.View ArticlePubMedGoogle Scholar
- Schleiffe E, Becker T: Common ground for protein translocation: access control for mitochondria and chloroplasts. Nat Rev Mol Cell Biol. 2011, 12: 48-59. 10.1038/nrm3027.View ArticleGoogle Scholar
- Searcy DG: Metabolic integration during the evolutionary origin of mitochondria. Cell Res. 2003, 13: 229-238. 10.1038/sj.cr.7290168.View ArticlePubMedGoogle Scholar
- Kleine T, Leister D: Retrograde signals galore. Front Plant Sci. 2013, 4: 1-3.View ArticleGoogle Scholar
- Stael S, Wurzinger B, Mair A, Mehlmer N, Vothknecht UC, Teige M: Plant organellar calcium signaling: an emerging field. J Exp Bot. 2012, 63: 1525-1542. 10.1093/jxb/err394.PubMed CentralView ArticlePubMedGoogle Scholar
- Clapham DE: Calcium signaling. Cell. 2007, 131: 1047-1058. 10.1016/j.cell.2007.11.028.View ArticlePubMedGoogle Scholar
- Tarasov AI, Griffiths EJ, Rutter GA: Regulation of ATP production by mitochondrial Ca + 2. Cell Calcium. 2012, 52: 28-35. 10.1016/j.ceca.2012.03.003.PubMed CentralView ArticlePubMedGoogle Scholar
- Alam MR, Groschner LN, Parichatikanond W, Kuo L, Bondarenko AI, Rost R, Waldeck-Weiermaier M, Mali R, Graier WF: Mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial Ca + 2 uniporter (MCU) contribute to metabolism secretion coupling in clonal pancreatic β-cells. J Biol Chem. 2012, 287: 34445-34454. 10.1074/jbc.M112.392084.PubMed CentralView ArticlePubMedGoogle Scholar
- Palty R, Silverman WF, Herschfinkel M, Caporale T, Sensi SL, Parnis J, Nolte C, Fishman D, Shoshan-Barmatz D, Herrmann S, Khananshvili D, Sekler I: NCLX is an essential component of mitochondrial Na+/Ca+2 exchange. Proc Natl Acad Sci U S A. 2010, 107: 436-441. 10.1073/pnas.0908099107.PubMed CentralView ArticlePubMedGoogle Scholar
- Mailloux RJ, Harper ME: Mitochondrial proticity and ROS signaling: lessons from the uncoupling proteins. Trends Endocrinol Metab. 2012, 23: 451-458. 10.1016/j.tem.2012.04.004.View ArticlePubMedGoogle Scholar
- Si Y, Palani S, Jayaraman A, Lee K: Effects of forced uncoupling protein1 expression in 3T3-L1 cells on mitochondrial function and lipid metabolism. J Lipid Res. 2007, 48: 826-836. 10.1194/jlr.M600343-JLR200.View ArticlePubMedGoogle Scholar
- Wang DY, Zhang Q, Liu Y, Lin ZF, Zhang SX, Sun MX, Sodmergen: The levels of male gametic mitochondrial DNA are highly regulated in angiosperms with regard to mitochondrial inheritance. Plant Cell. 2010, 22: 2402-2416. 10.1105/tpc.109.071902.PubMed CentralView ArticlePubMedGoogle Scholar
- Yoo SD, Cho YH, Sheen J: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2007, 2: 1565-1572. 10.1038/nprot.2007.199.View ArticlePubMedGoogle Scholar
- Ogawa K, Hatano-Iwasaki A, Yanagida M, Iwabuchi M: Level of glutathione is regulated by ATP-dependent ligation of glutamate and cysteine through photosynthesis in Arabidopsis thaliana: mechanism of strong interaction of light intensity with flowering. Plant Cell Physiol. 2004, 45: 1-8. 10.1093/pcp/pch008.View ArticlePubMedGoogle Scholar
- Keech O, Dizengremel P, Gardestrom P: Preparation of leaf mitochondria from Arabidopsis thaliana. Physiol Plant. 2005, 124: 403-409. 10.1111/j.1399-3054.2005.00521.x.View ArticleGoogle Scholar
- Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A: Full-length transcriptome assembly from RNA-seq data without reference genome. Nat Biotechnol. 2011, 29: 644-652. 10.1038/nbt.1883.PubMed CentralView ArticlePubMedGoogle Scholar
- Wagner GP, Kin K, Lynch VJ: Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci. 2012, 131: 281-285. 10.1007/s12064-012-0162-3.View ArticlePubMedGoogle Scholar
- Langmead B, Trapnell C, Pop M, Salzberg SL: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009, 10: R25-10.1186/gb-2009-10-3-r25.PubMed CentralView ArticlePubMedGoogle Scholar
- Tomato Genome Consortium: The tomato genome sequence provides insights into fleshy fruit evolution. Nature. 2012, 485: 635-641. 10.1038/nature11119.View ArticleGoogle Scholar
- Naika M, Shameer K, Mathew OK, Gowda R, Sowdhamini R: STIFDB2: An updated version of plant stress-responsive transcription factor database with additional stress signals, stress-responsive transcription factor binding sites and stress-responsive genes in Arabidopsis and rice. Plant Cell Physiol. 2013, 54: 1-15. 10.1093/pcp/pcs172.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.