Article Figures & Data
Figures
- Figure 1.
General B. sinuspersici and S. aralocaspica Chlorenchyma Cell Anatomy and in Situ Immunolocalization of Rubisco, PPDK, and NADP-MDH.
(A) Cross section of a Bienertia leaf showing a chlorenchyma cell containing two cytoplasmic compartments, a CCC and a PCC. Cytoplasmic channels connect the two compartments (small arrows).
(B) Cross section of an S. aralocaspica leaf showing chlorenchyma cells with organelles compartmentalized in the distal (D) and proximal (P) regions.
(C) In Bienertia, Rubisco is localized mainly in the chloroplasts of the CCC.
(D) In S. aralocaspica, Rubisco is localized in the chloroplasts of the proximal compartment.
(E) In Bienertia, PPDK is highly concentrated in the chloroplasts in the PCC, with lower levels in the CCC.
(F) In S. aralocaspica, PPDK is strongly localized to chloroplasts in the distal compartment.
(G) In Bienertia, NADP-MDH is localized mainly in the chloroplasts of the peripheral compartment.
(H) In S. aralocaspica, NADP-MDH is localized mainly in the chloroplasts of the distal compartment.
N, nucleus. Bars = 20 μm.
- Figure 2.
B. sinuspersici and S. aralocaspica Belong to the NAD-ME Group of C4 Plants.
Immunoblot showing the reactivity of Bienertia and S. aralocaspica total proteins to C4 enzymes and Rubisco. Protein blots were probed with polyclonal antibodies raised against Z. mays PEPC and PPDK, Amaranthus hydrochondriacus NAD-ME, and Spinacea oleracea Rubisco large subunit. Numbers at left indicate molecular mass standards in kilodaltons.
- Figure 3.
Cell-Permeant Fluorescent Staining and Immunofluorescence Labeling Showing the Distribution of ER, Mitochondria, Nuclei, Peroxisomes, and Vacuoles in B. sinuspersici and S. aralocaspica Chlorenchyma Cells.
Representative results from three separate experiments are shown. Confocal microscopy of live and fixed Bienertia ([A] to [E]) and S. aralocaspica ([F] to [J]) chlorenchyma cells stained with various organelle-specific fluorescent dyes and antibodies demonstrate their spatial relationship with chloroplasts (red). Bars = 10 μm.
(A) and (F) Projections of chlorenchyma cells stained with DiOC6(3) showing reticular structures (green).
(B) and (G) Projections of rhodamine 123–stained chlorenchyma cells showing the concentration of mitochondria (yellow-green) in the CCC and proximal compartment.
(C) and (H) Single optical section (C) and projection of acridine orange–stained cells (H) showing the prominent position of nuclei (N; green) relative to the CCC and proximal compartment.
(D) and (I) Projections of fixed cells probed with catalase and Oregon green–conjugated secondary antibodies to visualize peroxisome (yellow-green) distribution.
(E) and (J) Single optical sections through the midplane of 5′(6)-carboxy-2′7′-dichlorofluorescein diacetate (carboxy-DCFDA)–stained cells showing the large vacuoles (green).
- Figure 4.
Peroxisomal Motility in B. sinuspersici Chlorenchyma Cells Transiently Expressing GFP-MFP.
Time-lapse images of a GFP-MFP–expressing B. sinuspersici chlorenchyma cell. The movement of three peroxisomes was monitored for 25 s. One of these peroxisomes (stars) showed oscillatory movement over the entire series. Another peroxisome (arrowheads) remained fixed at a site for 5 s and then exhibited short-distance movement during the last 20 s. A third peroxisome (arrows) demonstrated continuous movement through the cytosol, covering a total distance of >40 μm. Numbers indicate elapsed time in seconds. Bar = 15 μm.
- Figure 5.
The Actin Cytoskeleton in Chlorenchyma Cells of B. sinuspersici and S. aralocaspica.
Immunofluorescence staining of actin demonstrates actin–chloroplast association in Bienertia and S. aralocaspica chlorenchyma cells. Actin filaments (green) were visualized with Oregon green–conjugated secondary antibody, and chloroplasts (red) were observed using their autofluorescence. Except for (A), (B), and (G), the images are single optical sections that demonstrate the direct interaction between actin filaments and chloroplasts. These images represent merged images of the dual channels that show their interaction. This is a representative result from at least five separate experiments with >50 cells observed. Arrows in (A) and (E) show the thick actin filament bundles connecting the CCC and the PCC. (G), (H), and (K) show that the nucleus (N) is also surrounded by actin filaments. Bars = 10 μm in (A), (B), (E), (G), (H), and (K) and 5 μm in (C), (D), (F), (I), (J), (L), and (M).
(A) and (G) Composite images (projections) of 30 optical 0.8-μm sections depicting the general actin filament patterns in Bienertia and S. aralocaspica chlorenchyma cells, respectively.
(B) Projection of a low-resolution image of the PCC showing the general distribution of the actin filaments.
(C) and (D) Single optical sections of high magnification of a region within the peripheral compartment demonstrating the close contact of actin filaments (arrows) with the chloroplasts.
(E) and (F) Single optical sections illustrating actin filaments surrounding and emanating from the nucleus (N).
(G) and (H) Projection (G) and single optical section (H) showing the two types of actin filaments: thick actin microfilament bundles (MFB) and thin actin microfilaments (MF) in S. aralocaspica.
(I) and (J) Single optical sections demonstrating the positioning of chloroplasts along the actin cables (arrows) by attaching to the thin actin filaments in the distal compartment.
(K) Optical section showing the actin filament pattern in the proximal compartment.
(L) and (M) Single optical sections of closeup images showing baskets of actin filaments (arrows) completely surrounding the chloroplasts.
- Figure 6.
The Microtubule Cytoskeleton in Chlorenchyma Cells of B. sinuspersici and S. aralocaspica.
Fixed chlorenchyma cells were labeled with anti-tubulin antiserum. Microtubules (green) were visualized with Oregon green–conjugated secondary antibody, the chloroplasts (red) were imaged using their autofluorescence, and the dual-wavelength confocal microscopic images were merged showing their association. This is a representative result obtained from at least five separate experiments with >50 cells observed. Bars = 10 μm in (A), (G), (L), and (M) and 5 μm in (B) to (F) and (H) to (K).
(A) and (G) Projections of a z-series of 30 optical 0.8-μm sections illustrating the overall microtubule patterns in Bienertia and S. aralocaspica chlorenchyma cells, respectively.
(B) Single optical section of the cortical region of a chlorenchyma cell to demonstrate an extensive network of microtubules.
(C) and (D) Single optical sections of closeup images showing a ring of microtubules (arrows) surrounding the peripheral chloroplasts.
(E) Single optical slice taken at the midpoint of the CCC surrounded by a thick cage of microtubules.
(F) Single optical section image of the cortical region showing microtubules surrounding the nucleus (N).
(H) Projection of the distal compartment showing a dense network of microtubules.
(I) Single optical section of the distal compartment showing transverse or oblique microtubules.
(J) and (K) Single optical sections of closeup images of chloroplasts in the distal region surrounded by rings of microtubules. Arrows indicate rings or baskets of microtubules around chloroplasts.
(L) Single optical section through the center of the proximal compartment showing the interaction of microtubules with the nucleus (N) and chloroplasts.
(M) Single optical section through the outer cortical region of the proximal compartment showing densely packed chloroplasts with a nucleus (N) among them and microtubules interweaving around these organelles.
- Figure 7.
Live Cell Localization of GFP Fusion Proteins.
B. sinuspersici chlorenchyma cells transiently expressing GFP chimeric proteins to actin binding protein (talin) or MAP4. Bars = 10 μm in (A) and (C) and 5 μm in (B) and inset in (C).
(A) Chlorenchyma cell transformed with GFP-talin showing thick actin filament bundles extending from the nucleus (N) and the CCC.
(B) Closeup image of a chlorenchyma cell transformed with GFP-talin showing actin filaments interacting with chloroplasts in the cortical region.
(C) Chlorenchyma cell transformed with GFP-MAP4 showing a dense network of microtubules in the cortical region. The inset shows an optical section through the cortical region of a GFP-MAP4–expressing cell showing both microtubules and chloroplasts.
- Figure 8.
Effects of CD and Ory on the Organization of the Central Cytoplasmic Compartment in B. sinuspersici Chlorenchyma Cells.
(A), (D), and (G) Autofluorescence of chloroplasts of chlorenchyma cells incubated over a 2-h period in stabilizing buffer (A) and in the same buffer containing 100 μM CD (D) or 30 μM Ory (G).
(B) and (C) Control chlorenchyma cells were fixed and probed with anti-actin (B) or anti-tubulin (C) antiserum to test the effects of the drugs on the two cytoskeletal elements.
(E) and (F) CD-treated cells probed with actin and tubulin antibodies showing complete disruption of actin filaments (E) with intact microtubules (F).
(H) and (I) Ory-treated chlorenchyma cells with dispersed CCC. The actin filaments remain intact (H), whereas microtubules are completely disrupted (I).
These are representative results from at least three separate experiments. Bars = 10 μm.
- Figure 9.
Effects of CD and Ory on the Distribution of Chloroplasts in the Two Cellular Compartments of S. aralocaspica Chlorenchyma Cells.
(A), (D), (G), and (J) Autofluorescence images of chlorenchyma cells incubated over a 2-h period in stabilizing buffer (A) and in the same buffer containing 100 μM CD (D), 30 μM Ory (G), or a combination of both drugs (J).
(B) and (C) Control chlorenchyma cells were fixed and probed with anti-actin (B) or anti-tubulin (C) antiserum to demonstrate the effectiveness of the drugs on the two cytoskeletal elements.
(E) and (F) CD-treated cells showing the complete disruption of actin filaments (E), but the transverse orientation of microtubules is not affected (F).
(H) and (I) Ory-treated chlorenchyma cells showing intact actin filaments (H) and complete disruption of microtubules (I).
(K) and (L) CD- and Ory-treated chlorenchyma cells showing complete disruption of both cytoskeleton systems and the aggregation of chloroplasts in the distal compartment.
These are representative results from at least three independent experiments. Bars = 10 μm.
Additional Files
Supplemental Data
Files in this Data Supplement:
- Supplemental Figure 1 - Detergent Resistant Components in Bienertia sinuspersici and Suaeda aralocaspica Chlorenchyma Cells. Scanning electron micrographs of Bienertia (A-C) and S. aralocaspica (D-G) chlorenchyma cells treated in detergent (Triton X-100) depicting detergent resistant components (arrowheads) associating with chloroplasts within the cells. Bars in A = 15 μm, D, E, and F = 5 μm, and B, C, and G = 1 μm.
- Supplemental Figure 2 - Specificity of the Anti-Actin and Anti-Tubulin Antibodies. Immunoblot analysis showing the specificity and reactivity of anti-actin and anti-β-tubulin antibodies with Bienertia and S. aralocaspica total protein. Ten μg of total protein from Bienertia (lanes 2 and 4) and S. aralocaspica (lanes 3 and 5) mature leaves were probed with antibodies raised against human skeletal muscle actin and rat brain β -tubulin. Numbers on the left indicate molecular mass in kDa (lane 1).
- Supplemental Figure 3 - The Actin and Microtubule Cytoskeleton in Chlorenchyma Cells of Suaeda heterophylla (C3) and Suaeda eltonica (C4 Kranz). Immunofluorescence staining of actin filaments (A, B, E, and F) and microtubules (C, D, G, and H) demonstrating actin- and microtubule-chloroplast association in S. heterophylla (A-D) and S. eltonica (E-H) chlorenchyma cells, respectively. Actin filaments and microtubules (green) were visualized with Oregon-green conjugated secondary antibody and chloroplasts (red) were observed using their autofluorescence. The images represent merged images of the dual channels which show their interaction. This is a representative result from at least three independent experiments with more than thirty cells observed. (A) and (C) Projections of low resolution images demonstrating the overall actin and microtubule cytoskeleton in mesophyll cells of S. heterophylla, respectively. (B) and (D) Optical sections of high magnification images showing the direct interaction between actin filaments and microtubules and chloroplasts in mesophyll cells of S. heterophylla. (E) and (G) Projections of low resolution images showing the actin and microtubule cytoskeleton in mesophyll cells of S. eltonica, respectively. (F) and (H) Projections of low resolution images showing the actin and microtubule cytoskeleton in bundle sheath cells of S. eltonica, respectively. The insets in (E) to (H) are high resolution optical sections showing the direct interaction between the cytoskeleton and chloroplasts in S. eltonica. Bars in A, C, E, F, and H = 10 μm; Bars in B, D, and insets = 5 μm; Bar in G = 15 μm.
- Supplemental Figure 4 - Effect of Cytochalasin D (CD) and Oryzalin (Ory) on Bienertia Chlorenchyma Cell Viability. Drug-treated chlorenchyma cells were rinsed in PME buffer containing 0.5 M mannitol and incubated in the same buffer containing the appropriate concentrations of rhodamine 123 and DIOC6(3) 24 h after removal of the drugs. (A), (B), and (C) Single optical sections through the mid section of control (A), CD- (B) or Ory-treated (C) chlorenchyma cells stained with rhodamine 123. Only control and CD-treated chlorenchyma cells showed the concentration of fluorescently stained mitochondria (yellow-green) in the CCC. Note the lack of CCC organization and staining in Ory-treated cells. (D), (E), and (F) Projections of control (D), CD- (E) or Ory-treated (F) chlorenchyma cells stained with DiOC6 (3). Staining of membranous structures (yellow-green) was observed only in control and CD-treated chlorenchyma cells. Note the absence of staining in Ory-treated cells. Bar = 20 μm.
