Assay of CFDA transport in the phloem cells of a detached <i>Arabidopsis</i> leaf.

<p>Leaves were observed by epifluorescence microscopy, under a fluorescent stereo microscope. The fluorescence appears as false color presentation using ImageJ’ ‘FIRE’ LUT. (a, b) Observation of fluorescence after the application of CFDA to a treated area of the leaf. (a) The veins in the loading area are already fluorescently labeled after 8 s. (b) After 180 s, the labeling extends to the main vein, secondary vein and minor veins around the loading area. (c-f) Observation of the transport of the fluorescent label after the application of CFDA to the treated area. (c) Observation of leaf autofluorescence immediately after application of the tracer. Treated areas are less fluorescent, enhancing observation of the vascular network (white arrows). For this experiment, the superficial layers were peeled away from a single vein in two areas: the loading area (la) and the transport area (ta). CFDA was loaded in the loading area. (d) 10 seconds after the application of CFDA. (e) 40 seconds after the application of CFDA, the fluorescence is transported and is visible in the transport area. (f) 100 seconds after the application of CFDA, the fluorescence has moved beyond the transport area. La: loading area; ta: transport area; mv: minor vein, sv: secondary vein; v: main vein. Scale bar = 5 mm.</p

Phloem markers available for the identification of phloem cells.

<p>Phloem markers available for the identification of phloem cells.</p

Imaging of subcellular compartments and new markers in sieve elements.

<p>Fluorescent proteins observed in leaves from plants carrying GFP expressed in different subcellular compartments. Images were obtained by CLSM. Fluorescence is shown in false color. (a) and (b) Observation of discrete bodies in a <i>pRTM1</i>:<i>GFP</i>:<i>RTM1</i> plant. The bodies in the sieve elements are up to 1 μm in diameter. In (h) colocalization of GFP:RTM1 with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (c) and (d) Observation of fluorescence in a <i>pRTM2</i>:<i>RTM2</i>:<i>GFP</i> plant. The signal is mostly localized at the vicinity of the plasma membrane, within the sieve element. It is also observed around spherical bodies (*) located next to the plasma membrane. In (d) colocalization of RTM2:GFP with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (e)–(i) Observation of PP2-A1:GFP in a <i>pSEOR2</i>:<i>PP2-A1</i>:<i>GFP</i> plant. PP2-A1 fluorescence indicates the presence of PP2-A1 in discrete spots located at the borders of the cell and in the vicinity of the sieve plate. In (f) and (g), details of the localization of PP2-A1:GFP around organelles present in the sieve elements. Arrows indicates circular structures surrounded by PP2-A1. In (h) colocalization of PP2-A1:GFP with MitoTracker Red. GFP fluorescence is shown in green and MitoTracker fluorescence is shown in red. In (i) colocalization of PP2-A1:GFP with mitochondria. GFP fluorescence is shown in green, chloroplast autofluorescence in red and MitoTracker fluorescence is shown in blue. Arrows indicate mitochondria surrounded by PP2-A1:GFP. mi: mitochondrion. pl: chloroplast. sp: sieve plate. lpb: large protein body. spb: small protein body. Scale bar = 5 μm.</p

Imaging of PP2-A1:GFP and free GFP in the phloem cells of a four-week-old leaf.

<p>Visualization of soluble GFP or PP2-A1 tagged with GFP or CFP in leaves from <i>pSUC2</i>:<i>PP2-A1</i>:<i>GFP, pSUC2</i>:<i>PP2-A1</i>:<i>GFP, pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> and <i>pSUC2</i>:<i>GFP</i> plants. Images were obtained by CLSM and fluorescence is shown in false colors. GFP or CFP signals are shown in green, and chlorophyll autofluorescence is shown in red. GFP-tagged PP2-A1 is found in the cytosol and nucleus of the companion cells. (a) Constructs for the imaging of fluorescence-tagged PP2-A1 and PP2-A2. (b) Observation, in a treated leaf area, of soluble GFP produced under the control of the <i>SUC2</i> promoter, in a <i>pSUC2</i>:<i>GFP</i> plant. GFP fluorescence is observed in veins of all orders (II, III and IV). (c) Localization of GFP fluorescence in a main vein from a <i>pSUC2</i>:<i>GFP</i> plant. The soluble GFP is found in companion cells and phloem parenchyma cells. (d) Localization of PP2-A1:GFP fluorescence to the junction of two veins from a <i>pSUC2</i>:<i>PP2-A1</i>:<i>GFP</i> plant. Bent companion cells (indicated by *) are typically found at such junctions. (e) Localization of PP2-A1:GFP in a vein from a <i>pSUC2</i>:<i>PP2-A1</i>:<i>GFP</i> plant. GFP is found only in the companion cells. This observation was made on a cross between the <i>pSUC2</i>:<i>PP2-A1</i>:<i>GFP</i> (in green) and <i>p35S</i>:<i>H2B</i>:<i>RFP</i> lines (in color red), so the nucleus is shown in yellow in the overlay, due to H2B:RFP and PP2-A1:GFP fluorescence. (f) Localization of GFP:PP2-A1 fluorescence in a minor vein from a <i>pSUC2</i>:<i>GFP</i>:<i>PP2-A1</i> plant. (g) Localization of <i>PP2-A1</i>:<i>CFP</i> fluorescence (shown in green) in a minor vein from a <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> plant. Typical distribution of plastids in the companion cells is observed. In the companion cells, the autofluorescent chloroplasts were aligned in a single file, whereas, in phloem parenchyma cells, the chloroplasts were located at the cell periphery. cc = companion cell; ppc = phloem parenchyma cell; se: sieve element. n: nucleus. Scale bar = b) 50 μm; (c)–(g) 10 μm.</p

Imaging of subcellular compartments in companion cells and phloem parenchyma cells.

<p>(a, c-h) Fluorescent proteins observed in leaves from plants resulting from crosses between <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> plants and lines carrying fluorescent proteins targeted to different subcellular compartments. Overlay images, obtained by CLSM, with fluorescence signals shown in false colors; CFP is shown in green (a, c-h) and GFP, YFP or RFP in red (a, c-h) except in (b). Superimposed pixels are shown in yellow. (a) Observation of chloroplasts in the companion cells of a <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> x <i>p35S</i>:<i>RbcS</i>:<i>YFP</i> plant (PP2-A1:CFP fluorescence is shown in green and RbcS:YFP in red). (b) Observation of the typical morphology of phloem cells of a <i>p35S</i>:<i>GFP</i>:<i>LTI6b</i> plant. GFP is shown in green and chloroplast autofluorescence is shown in red. Two types of phloem cells—companion cells and phloem parenchyma cells—were identified on the basis of plastid distribution (autofluorescence, shown in red). In these plants, GFP:LTI6b fluorescence (in green) could also be used to identify sieve elements. Phloem parenchyma cells are the largest cells and are located on the edge of the vasculature. Sieve elements lack chloroplasts. Companion cells display typical chloroplast alignments. (c) Observation of nuclei in the companion cells of a <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> x <i>p35S</i>:<i>H2B</i>:<i>RFP</i> plant. Companion cells have square-like nuclei. (d) Observation of the endoplasmic reticulum in the companion cells of a <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> x <i>p35S</i>:<i>ER</i>:<i>YFP</i> plant. In companion cells, the ER is found principally next to the plasma membrane and around the nucleus. On this image, the ER can also be seen in a sieve element aligned between two arrays of companion cells. (e) Observation of mitochondria in the companion cells of a <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> x <i>p35S</i>:<i>COX4</i>:<i>GFP</i> plant. On this image, large numbers of mitochondria can be seen in the companion cells. (f) Observation of vacuoles in the companion cells of a <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP x p35S</i>:<i>yTIP</i>:<i>YFP</i> plant. The image shows several vacuoles per companion cell. (g) Observation of actin network in the companion cells of a <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> x <i>p35S</i>:<i>FABD2</i>:<i>GFP</i> plant. Thick actin bundles can be seen whereas thin actin filaments are barely detectable. (h) Observation of cortical microtubules in bent companion cells at a vein junction in a <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> x <i>p35S</i>:<i>GFP</i>:<i>MBD</i> plant. Stars indicate the vacuoles. Scale bar = 5 μm.</p

Imaging of PP2-A1:GFP in sieve elements undergoing differentiation.

<p>Fluorescent PP2-A1:GFP protein observed in leaves of <i>pSEOR2</i>:<i>PP2-A1</i>:<i>GFP</i> plants. Images were obtained by CLSM. GFP fluorescence is shown in false color green, propidium iodide in nuclei is shown in red and plastid autofluorescence in magenta except in (e). (a) General overview of a main vein showing mature and immature sieve elements. (b) Immature sieve element with soluble PP2-A1:GFP. In this cell, a number of subcellular compartments, including organelles and the nucleus, are still present. (c) Immature sieve element with soluble PP2-A1:GFP, in which the nucleus is no longer observed, although plastid autofluorescence is still present. Arrows indicate plastids in an immature sieve element. (d) In some cells that have begun to differentiate, PP2-A1:GFP fluorescence aggregates around small organelles. As sieve elements mature, their organelles gradually disappear. PP2-A1:GFP is present in an aggregated form around organelles, presumably mitochondria, although still present in a soluble form in the cytosol. Arrows indicate dense PP2-A1 material, presumably around mitochondria and plastids. (e) In sieve element ongoing differentiation, PP2-A1:GFP fluorescence, shown in green, aggregates around mitochondria stained with MitoTracker (shown in red). Arrows indicate mitochondria. (f) In mature sieve elements, PP2-A1:GFP is present only attached to organelles. * indicates nuclei. Se: sieve element; i-se: immature sieve element; companion cell. Scale bar = 5 μm.</p

Imaging of subcellular compartments and known protein bodies in sieve elements.

<p>Fluorescent proteins observed in leaves from plants carrying GFP expressed in different subcellular compartments. Images were obtained by CLSM. Fluorescence is shown in false color. (a) Observation of the endoplasmic reticulum in the companion cells and the sieve elements of a <i>p35S</i>:<i>ER</i>:<i>YFP</i> x <i>pSUC2</i>:<i>PP2-A1</i>:<i>GFP</i> plant (same section as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118122#pone.0118122.g003" target="_blank">Fig. 3D</a>). YFP fluorescence is shown in yellow and GFP is shown in blue. (b) Observation of mitochondria with MitoTracker fluorescent dye, presented in false colors (green). Plastid autofluorescence is shown in red. Mitochondria are found in both the companion cells and sieve elements. (c) Observation of mitochondria with MitoTracker fluorescent dye, presented in false colors (blue) in the phloem of <i>p35S</i>:<i>COX4</i>:<i>GFP</i> plant (in yellow). Plastid autofluorescence is shown in red. Co-labeling of mitochondria with MitoTracker and COX4:GFP, in both the companion cells and sieve elements, is shown in green. Arrows indicate mitochondria in the sieve elements.(d), (e) and (f) Observation of the P-proteins in the sieve elements of a <i>pSEOR2</i>:<i>SEOR2</i>:<i>GFP</i> plant. In (d) and (e), GFP fluorescence is shown in green, plastid autofluorescence in red. In these images, the P-proteins form a typical plug (arrow) next to the sieve plate (in (c)), and discrete filaments in the lumen of the sieve element and protein agglomerates (in (d)). In (f), GFP fluorescence is shown in yellow, plastid autofluorescence in red, and MitoTracker fluorescence in blue.</p

Imaging of phloem markers in all orders of veins.

<p>Fluorescent proteins observed in leaves from plants carrying GFP or CFP fused to different proteins, either in the companion cells, or in the sieve elements. Images were obtained by CLSM and luorescence is shown in false color, with GFP shown in green, CFP shown in blue and plastid autofluorescence shown in red. Left panel: main vein. Middle panel: secondary vein. Right panel: minor vein. (a) Observation of <i>pSUC2</i>:<i>PP2-A1</i>:<i>GFP</i> plant. (b) Observation of <i>pSUC2</i>:<i>PP2-A1</i>:<i>CFP</i> plant. (c) Observation of <i>pSEOR2</i>:<i>PP2-A1</i>:<i>GFP</i> plant. (d) Observation of <i>pSEOR2</i>:<i>SEOR2</i>:<i>GFP</i> plant. (e) Observation of <i>pRTM1</i>:<i>GFP</i>:<i>RTM1</i> plant. Scale bar = 20 μm.</p

Q-RT-PCR analysis of the expression level of the three <i>RTM</i> genes in different Arabidopsis accessions.

<p>(<b>A</b>) <i>RTM1</i> expression; (<b>B</b>) <i>RTM2</i> expression; (<b>C</b>) <i>RTM3</i> expression. Fold change is determined relative to the value of Col-0 which is set arbitrarily at 1. The qPCR results are normalized to an ubiquitine-conjugating enzyme family gene (At2g36060). The graph represents the average values from three independent experiments involving 3 plants each. Bars represent SD of Ct values calculated using the Roche software. * : P<0.05; indicates that scoring values differ significantly from Col-0. Nd: not determined.</p

RTM allelic pattern and infection phenotype with LMV isolates of each Arabidopsis accession.

a<p>Numbers in each column corresponding to each <i>RTM</i> allele refer to the <i>RTM</i> allele numbers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039169#pone-0039169-g001" target="_blank">Figure 1</a>. The non-functional alleles are in bold.</p>b<p>R: resistant to LMV systemic infection; S: susceptible to LMV systemic infection; - : not determined.</p