Article Figures & Data
Figures
- Figure 1.
MDP25 Directly Binds to Microtubules in Vitro.
(A) GST-MDP25 fusion protein was cosedimented with paclitaxel-stabilized microtubules. GST-MDP25 mostly appeared in the supernatant in the absence of microtubules (S) but cosedimented with microtubules into the pellets (P).
(B) The results of the quantitative analysis of the binding between GST-MDP25 and microtubules are shown. The amount of protein was determined by gel scanning.
(C) to (K) Immunofluorescence staining was performed to investigate the binding of MDP25 to microtubules in vitro. Red fluorescence signals indicate rhodamine-labeled microtubules, and green signals indicate GST-MDP25 probed with anti-GST and fluorescein isothiocyanate–conjugated secondary antibodies. Microtubules (C), MDP25 (D), and merged image (E). Microtubules (F), denatured MDP25 (G), and merged image (H). Microtubules (I), GST (J), and merged image (K). Notice that the microtubules exhibited a single-filament pattern, and no microtubule bundles were observed in the presence of MDP25 fusion protein.
(L) to (N) The plasma membrane localization of MDP25 occurred independently of cortical microtubules. Seedlings of PMDP25:MDP25:GFP were treated with 10 μM oryzalin for 60 min. P35S:GFP (L), PMDP25:MDP25:GFP (M), and seedlings of (M) treated with oryzalin (N). The GFP fluorescence intensity was averaged from the measurements of at least 14 cells after background subtraction, and the result in (N) is expressed as the ratio of fluorescence intensity of treated cells to untreated cells.
Bars = 10 μm in (K) and (N).
- Figure 2.
MDP25 Inhibits Tubulin Assembly in Vitro.
(A) Various concentrations of GST-MDP25 were added to a 30 μM tubulin solution, and turbidity was monitored during polymerization. The tubulin polymerization rate and mass of the microtubules at a steady state of tubulin polymerization decreased in a concentration-dependent manner when added to MDP25. The addition of 8 μM GST did not affect the mass of microtubules in a steady state of tubulin assembly.
(B) to (D) Microtubules were polymerized in a 20 μM rhodamine-labeled tubulin solution in the absence of GST-MDP25 (B), in the presence of 8 μM GST-MDP25 (C), and in the presence of 10 μM GST (D). Bar in (D) = 10 μm for (B) to (D).
- Figure 3.
MDP25-Dependent Microtubule Depolymerization in Vitro.
(A) and (C) MDP25 directly induced microtubule depolymerization in a TIRFM assay. Preformed rhodamine-labeled microtubules were incubated with 80 nM MDP25 (A) or 0 nM MDP25 (C), and the images of immobilized microtubules were recorded for 10 min at 10-s intervals. A few fluorescent images at different time points during the recording process are shown.
(B) and (D) Kymographs show the effect of preformed microtubules depolymerized by MDP25 or without MDP25, respectively.
(E) An analysis summary of at least 30 microtubules of each sample shows the depolymerization rate at both microtubule ends.
Horizontal bars = 5 μm; vertical bars = 2 min. Error bars indicate ± sd.
- Figure 4.
MDP25 Negatively Regulates Hypocotyl Cell Elongation.
(A) Physical structure of Arabidopsis MDP25. MDP25 contains four exons and three introns, which are represented by filled boxes and lines, respectively. The position of a T-DNA insertion mutant, designated mdp25 (T-DNA line SALK_241_A08), is noted by arrows above the diagram.
(B) RT-PCR analysis of full-length transcripts of MDP25 in seedlings of the wild-type Columbia ecotype (WT), MDP25-GFP transgenic Arabidopsis line (OE), and mdp25 mutant, with 18S rRNA used as a control.
(C) and (D) The mdp25 mutant has longer hypocotyls (t test, *P < 0.05), whereas the hypocotyls are shorter in MDP25-GFP transgenic Arabidopsis grown on half-strength Murashige and Skoog in continuous light for 7 d ([C]; t test, **P < 0.01) or the dark (D) for 6 d. The graph shows the average hypocotyl length measured from at least 22 seedlings under light growth. Error bars indicate ±se.
(E) Shapes of hypocotyl cells were disordered in MDP25-GFP transgenic Arabidopsis and mdp25 seedlings. The top panels show the scanning electron microscopy images, and the bottom panels show the light microscopy images of hypocotyl cross sections.
(F) to (I) MDP25 was mainly expressed in the nongrowing region of dark-grown hypocotyls of Arabidopsis.
(F) Histochemical GUS staining of PMDP25:GUS:TMDP25 transgenic seedlings grown in the dark for 3, 4, and 5 d.
(G) Confocal images of hypocotyls of 4-d-old dark-grown PMDP25:MDP25:GFP transgenic plants. The epidermal cells of a cell file of the hypocotyl were numbered from the base to the top. “Base” represents the region of 1 to 10 cells, and “Top” represents the region of 11 to 20 cells.
(H) and (I) RT-PCR (H) and immunoblot (I) against anti-GFP antibody show that MDP25 was highly expressed in the basal region and minimally expressed in the top region of etiolated hypocotyls. 18S rRNA or actin was used as a loading control. Three biological replicates were performed and led to similar results.
Bars = 100 μm in (E) and (G).
[See online article for color version of this figure.]
- Figure 5.
The Cortical Microtubule Array Is Greatly Altered in Etiolated Epidermal Hypocotyl Cells of MDP25-Overexpressing Seedlings.
(A) Cortical microtubules in etiolated hypocotyl epidermal cells of MDP25-overexpressing (OE) and mdp25 seedlings with a yellow fluorescent protein–tubulin background in different regions (upper hypocotyl region, middle hypocotyl region, and basal cells) were observed by confocal microscopy after growth in the dark for 72 h. WT, wild type. Bar = 10 μm.
(B) Frequency of microtubule orientation patterns in different regions of etiolated hypocotyl epidermal cells of the wild type, OE, and the mdp25 mutant (n > 90 cells).
[See online article for color version of this figure.]
- Figure 6.
Cortical Microtubules Are Hypersensitive in MDP25-Overexpressing Arabidopsis Cells but More Resistant in mdp25 Mutant Cells to Oryzalin Treatment.
(A) to (D) Cortical microtubules were observed in epidermal cells in the basal region of etiolated hypocotyls in wild-type (WT), MDP25-overexpressing (OE), and mdp25 mutant seedlings after treatment with 0 μM oryzalin (A), 5 μM oryzalin for 5 min (B), 5 μM oryzalin for 10 min (C), and 10 μM oryzalin for 30 min (D).
(E) After the treatment in (D), oryzalin was washed off, and cortical microtubules were imaged 2 h later. Bar = 10 μm.
(F) Quantification of cortical microtubules in hypocotyl epidermal cells of the wild type, OE, and mdp25 mutant by ImageJ software (n > 50 cells of each sample). Vertical scale represents the number of cortical microtubules across a fixed line (~10 μm) vertical to the orientation of most cortical microtubules in the cell. t tests were performed to compare the number of cortical microtubules in hypocotyl epidermal cells of OE and mdp25 with that of the wild type at the same conditions. **P < 0.01 and * P < 0.05 by t test. Error bars represent ±sd.
- Figure 7.
Residues 1 to 23 of MDP25 Are Essential for Targeting MDP25 to Microtubules.
(A) Residue conservation and variability of the MDP25 DREPP domain. The conservation score and variability coefficient were averaged over a sliding window of 11 residues, which are shown in the panel as a green curve relating to the left y axis and red curve relating to the right y axis. The position of the conserved region (i.e., residues with higher conservation scores and lower variability coefficients) is labeled.
(B) The conserved domain of MDP25 across plant species is responsible for microtubule (MT) binding. GST-MDP25 1–132 but not GST-MDP25 133–225 cosedimented with paclitaxel-stabilized microtubules. P, pellet; S, supernatant.
(C) Alignment of amino acid sequences 1 to 132 of MDP25 with MAP18. The asterisks show the sites in the MDP25 amino acid sequence that were mutated (Lys to Ala).
(D) GST-MDP25 24–132 did not cosediment with paclitaxel-stabilized microtubules.
(E) The amount of MDP25 24–132 was estimated following gel density scanning in the absence or presence of 5 μM microtubules from three independent experiments. The amount of MDP25 24–132 in the pellet is expressed as a percentage of total MDP25 24–132.
(F) Compared with wild-type MDP25, less mutated MDP25 fusion protein cosedimented with paclitaxel-stabilized microtubules.
(G) The results of the quantitative analysis of the amount of GST-MDP25 fusion protein in the pellets are shown. The amount of protein was determined by gel scanning from three independent experiments (means ± sd, n = 3).
(H) to (K) Microtubules were polymerized in a 20 μM rhodamine-labeled tubulin solution in the absence of GST-MDP25 (H), in the presence of 8 μM GST-MDP25 (I), in the presence of 10 μM MDP25 K7A (J), or in the presence of 10 μM MDP25 K18A (K). Bar in (K) =10 μm for (H) to (K).
[See online article for color version of this figure.]
- Figure 8.
Calcium Partially Dissociates MDP25 from the Plasma Membrane to the Cytosol.
(A) MDP25-GFP transgenic suspension cells were treated with A23187 with or without free Ca2+ for 1, 2, 3, or 4 h. Bar = 20 μm.
(B) GFP signals of MDP25 protein were quantified and are expressed as the ratio of the average fluorescence intensity of the cytosol in each cell. The data represent the means ± sd of three independent experiments; 22 suspension cells were analyzed per sample.
(C) The blot assay showed that the amount of MDP25 protein increased in the soluble fraction when the cells were treated with A23187 plus Ca2+ for 2 and 4 h. Lane 1, wild-type cells; lanes 2 to 6, MDP25-GFP transgenic cells.
[See online article for color version of this figure.]
- Figure 9.
Microtubule-Destabilizing Activity of MDP25 Is Enhanced in Cells When Cytoplasmic Calcium Levels Are Elevated.
Cortical microtubules are supersensitive to oryzalin after treatment with A23187 plus Ca2+ in MDP25-overexpressing suspension cells. MDP25-overexpressing cells (OE) ([A] and [D]), wild-type cells (WT) ([B] and [E]), mdp25 cells ([C] and [F]) in the absence of oryzalin ([A] to [C]; −Oryzalin) for 15 min and presence of oryzalin ([D] to [F]; +Oryzalin).
(A) to (C) The fluorescence images showed a parallel array of cortical microtubules in the presence or absence of A23187 or Ca2+ in MDP25-overexpressing, wild-type, and mdp25 suspension cells, but most cortical microtubules were disordered in the presence of A23187 plus Ca2+ in MDP25-overexpressing cells.
(D) Most cortical microtubules were disrupted in MDP25-overepxressing cells pretreated with A23187 plus Ca2+, but most were relatively normal in the presence of A23187 or Ca2+ alone with 1 μM oryzalin.
(E) and (F) However, in wild-type and mdp25 suspension cells, cortical microtubules showed little difference in sensitivity to oryzalin treatment when pretreated with A23187, Ca2+, or A23187 plus Ca2+. Compared with cortical microtubules in wild-type cells, cortical microtubules in mdp25 cells were less sensitive to oryzalin treatment when pretreated with A23187 plus Ca2+. Bar = 20 μm in (F).
(G) Quantification of cortical microtubules in Arabidopsis suspension cells of wild-type, MDP25-overexpressing, and mdp25 mutant cells by ImageJ software (n > 50 cells for each sample). Vertical scale represents the number of cortical microtubules across a fixed line (~10 μm) that is vertical to the orientation of most cortical microtubules in the cell. t tests were performed to compare the number of cortical microtubules in suspension cells of OE and mdp25 with that of the wild type at the same conditions. Error bars represent ±sd; **P < 0.01 and *P < 0.05 by t test.
[See online article for color version of this figure.]
- Figure 10.
Model of MDP25 Function on Cortical Microtubules in the Mediation of Cell Elongation.
During the resting state cytosolic calcium level, MDP25 dominantly binds to the plasma membrane, and its microtubule binding domain is covered in phosphatidylinositol phosphates. Increased cytosolic calcium levels induce partial MDP25 disassociation from the plasma membrane. Increased cytosolic MDP25 levels destabilize cortical microtubules (MTs) by depolymerizing microtubules, result in the disorganization of cortical microtubules, and further inhibit cell elongation.
Tables
- Table 1.
Size of Hypocotyl Cells of MDP25 Transgenic Arabidopsis Grown in the Light for 7 d
Parameter of Hypocotyl Cells Wild Type MDP25 Overexpressing mdp25 Length of hypocotyl cortex cells (μm) 124.68 ± 34.55 (n = 100) 60.80 ± 10.01 (n = 110) 145.61 ± 29.14 (n = 100) Width of hypocotyl cortex cells (μm) 28.55 ± 5.88 (n = 100) 33.28 ± 5.50 (n = 110) 28.32 ± 4.82 (n = 100)
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