Cold Treatment Induces Transient Mitochondrial Fragmentation in Arabidopsis thaliana in a Way that Requires DRP3A but not ELM1 or an ELM1-Like Homologue, ELM2
"> Figure 1
<p>Mitochondrial morphologies in wild-type Arabidopsis and the <span class="html-italic">elm1-1</span> mutant. The images show GFP-labeled mitochondria in leaf epidermal cells. Mitochondria in the <span class="html-italic">elm1-1</span> mutant are longer and fewer than those in the wild type, because of the disturbance of mitochondrial fission in the mutant. However, <span class="html-italic">elm1-1</span> cells still have many short mitochondria (arrows), suggesting that mitochondrial fission can occur without ELM1. Scale bar, 10 μm, is applicable to the both figures.</p> "> Figure 2
<p><span class="html-italic">ELM2</span> encodes an ELM1-like protein and GFP-tagged ELM2, like ELM1, localizes to the mitochondrial surface. (<b>a</b>) Clustal W alignment of ELM1 and ELM2 amino acids sequences. * depicts the positions of numbers in every ten amino acids. (<b>b</b>) Localization of GFP-ELM2 surrounding mitochondria. Arabidopsis cultured cells transiently expressing GFP-ELM2 with a mitochondrial marker MitoTracker were examined by confocal laser scanning microscopy (CLSM). A part of a single cell is shown. <span class="html-italic">Left</span> and <span class="html-italic">Center</span> are separate images obtained with the GFP and MitoTracker, respectively. <span class="html-italic">Right</span> is the merged image. Scale bar, 5 μm, is applicable to the other two figures. Upper right insets are X2 enlarged images.</p> "> Figure 3
<p>Disruption of <span class="html-italic">ELM2</span> does not appear to affect mitochondrial morphology much. (<b>a</b>) A T-DNA insertion in the end of the 3rd intron in the <span class="html-italic">elm2</span> mutant. (<b>b</b>) RT-PCR of full length of <span class="html-italic">ELM2</span> ORF (open reading frame) in the wild-type, <span class="html-italic">elm1-1</span>, <span class="html-italic">elm2</span> and <span class="html-italic">elm1-1 elm2</span> double mutants. (<b>c</b>) Comparison of growing phenotypes of wild-type, <span class="html-italic">elm1-1</span>, <span class="html-italic">elm2</span> and <span class="html-italic">elm1-1 elm2</span> double mutants. 30-day-old plants. Scale bar, 5 cm. (<b>d</b>) Mitochondrial morphologies in the wild-type, <span class="html-italic">elm1-1</span>, <span class="html-italic">elm2</span> and <span class="html-italic">elm1-1 elm2</span> double mutants. Leaf epidermal cells in 14-day-old plants were observed by confocal laser scanning microscopy. Scale bar, 10 μm, is applicable to the four images. (<b>e</b>) Average planar areas of mitochondria of wild type and mutants. (<span class="html-italic">n</span> > 218 in each of three replications) in each mutant. Error bars show S.E. ** indicates statistical significance at <span class="html-italic">p</span> < 0.01.</p> "> Figure 4
<p>Heterologous complementation test of mitochondrial morphology in the <span class="html-italic">elm1</span> mutant by expression of <span class="html-italic">ELM2</span>. (<b>a</b>) Schematic drawing of DNA constructs used in this study. <span class="html-italic">ELM1</span>, <span class="html-italic">ELM2</span> and <span class="html-italic">GUS</span> coding sequences are attached between the probable promoter, the 950bp upstream region of <span class="html-italic">ELM1</span> and the sequence of CaMV35S terminator. (<b>b</b>) RT-PCR of the full length of the <span class="html-italic">ELM2</span> ORF in the wild-type, <span class="html-italic">elm1-1</span>, and three <span class="html-italic">elm1-1</span> mutants transformed with <span class="html-italic">ELM1pro:ELM1</span>, <span class="html-italic">ELM1pro:ELM2</span> and <span class="html-italic">ELM1pro: GUS</span> respectively. (<b>c</b>) Occurrence of elongate mitochondria in leaf epidermal cells in 14-day-old cotyledons from five Arabidopsis lines (wild-type, <span class="html-italic">elm1-1</span>, and three <span class="html-italic">elm1-1</span> mutants transformed with <span class="html-italic">ELM1pro:ELM1</span>, <span class="html-italic">ELM1pro:ELM2</span> and <span class="html-italic">ELM1pro: GUS</span>). Occurrence is expressed as the percentage of 40 confocal laser scanning microscopic images obtained from 8 leaves from each line that were judged to have elongated mitochondria (as in the <span class="html-italic">elm1-1</span> image in <a href="#ijms-18-02161-f001" class="html-fig">Figure 1</a>). The experiments were repeated three times independently and the results were averaged. Error bars show S.E.</p> "> Figure 5
<p>Mitochondrial fragmentation was induced by cold treatment in the wild-type and <span class="html-italic">elm</span> mutants but not in the <span class="html-italic">drp3a-1</span> mutant. (<b>a</b>,<b>b</b>), Representative mitochondrial morphologies in the wild type and mutants at room temperature before and 1 h after 4 °C treatment. Each scale bar is applicable to the all images in (<b>a</b>,<b>b</b>), respectively. (<b>c</b>,<b>d</b>) Average planar areas of mitochondria in epidermal cells of wild type and mutants before (red bars) and 1 h after (blue bars) cold-temperature treatment (<span class="html-italic">n</span> > 218 in each of three replications). (<b>e</b>,<b>f</b>) Average number of mitochondria per 100 μm<sup>2</sup> in leaf epidermal cells of wild-type and mutants before (red bars) and 1 h after (blue bars) cold treatment. <span class="html-italic">n</span> = 3 Error bars show S.E. ** indicates statistical significance at <span class="html-italic">p</span> < 0.01 and * at <span class="html-italic">p</span> < 0.05. Because data sets (<b>a</b>,<b>c</b>,<b>e</b>) and (<b>b</b>,<b>d</b>,<b>f</b>) were collected independently in different conditions (e.g., laser strength, detector gain, etc.), they could not be compared with each other directly.</p> "> Figure 6
<p>Mitochondrial morphology in the wild type and <span class="html-italic">elm</span> mutants after different durations of cold treatment. Mitochondria were observed in leaf epidermal cells of 28-day-old plants grown at 22 °C before and after different durations of 4 °C treatment. Scale bar, 10 μm, is applicable to the all images.</p> "> Figure 7
<p>Time course observations of mitochondria and DRP3A in the <span class="html-italic">elm1</span> mutant. Images show a double-stained leaf epidermal cell of a 30-day-old <span class="html-italic">elm1-6</span> Arabidopsis plant transformed with DRP3Apro:DRP3A: GFP at different times after cold treatment. Bottom panels, mitochondrial network stained with MitoTracker; middle panels, DRP3AGFP; top panels, merged MitoTracker and GFP images. Cytosolic DRP3A: GFP first appeared as a hazy signal (0 and 30 min) and gradually localized and concentrated on mitochondria (40, 50 and 110 min). Arrows indicate sites of mitochondrial fission. Scale bar, 10 μm, is applicable to the all images.</p> "> Figure 8
<p>Schematic model of Arabidopsis mitochondrial fission. Two types of mitochondrial fission are drawn. In the normal condition (left, shown as RT (room temperature) 22 °C), the division executor, DRP3 localizes to mitochondria via interaction with ELM1. In the case of mitochondrial fission transiently induced by cold treatment, DRP3 could localize to mitochondria by skipping the help of ELM1 (and ELM2) and underwent fission.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Residual Mitochondrial Fission in the Mitochondrial Fission Mutant Elm1
2.2. Is Mitochondrial Fission without ELM1 due to ELM2?
2.3. Transient Mitochondrial Fragmentation by Cold Treatment
2.4. DRP3A Could Localize to Mitochondria without ELM1 at the Cold Treatment
3. Discussion
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
4.2. Construction of Plasmids
4.3. Agrobacterium Mediated Transformation of Arabidopsis Plants and Cultured Cells
4.4. MitoTracker Orange Staining
4.5. Microscopic Observations and Image Analysis
4.6. RT-PCR Analysis
4.7. Cold Treatment
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
DRP | Dynamin-related protein |
ELM1 | Elongate mitochondria |
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Arimura, S.-i.; Kurisu, R.; Sugaya, H.; Kadoya, N.; Tsutsumi, N. Cold Treatment Induces Transient Mitochondrial Fragmentation in Arabidopsis thaliana in a Way that Requires DRP3A but not ELM1 or an ELM1-Like Homologue, ELM2. Int. J. Mol. Sci. 2017, 18, 2161. https://doi.org/10.3390/ijms18102161
Arimura S-i, Kurisu R, Sugaya H, Kadoya N, Tsutsumi N. Cold Treatment Induces Transient Mitochondrial Fragmentation in Arabidopsis thaliana in a Way that Requires DRP3A but not ELM1 or an ELM1-Like Homologue, ELM2. International Journal of Molecular Sciences. 2017; 18(10):2161. https://doi.org/10.3390/ijms18102161
Chicago/Turabian StyleArimura, Shin-ichi, Rina Kurisu, Hajime Sugaya, Naoki Kadoya, and Nobuhiro Tsutsumi. 2017. "Cold Treatment Induces Transient Mitochondrial Fragmentation in Arabidopsis thaliana in a Way that Requires DRP3A but not ELM1 or an ELM1-Like Homologue, ELM2" International Journal of Molecular Sciences 18, no. 10: 2161. https://doi.org/10.3390/ijms18102161