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Live imaging of companion cells and sieve elements in Arabidopsis leaves.

Cayla T, Batailler B, Le Hir R, Revers F, Anstead JA, Thompson GA, Grandjean O, Dinant S - PLoS ONE (2015)

Bottom Line: The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix.GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids.The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institut Jean-Pierre Bourgin, INRA-AgroParisTech, UMR1318, ERL CNRS 3559, Saclay Plant Sciences, Versailles, France.

ABSTRACT
The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

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Imaging of subcellular compartments and known protein bodies in sieve elements.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 p35S:ER:YFP x pSUC2:PP2-A1:GFP plant (same section as in Fig. 3D). 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 p35S:COX4:GFP 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 pSEOR2:SEOR2:GFP 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.
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pone.0118122.g004: Imaging of subcellular compartments and known protein bodies in sieve elements.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 p35S:ER:YFP x pSUC2:PP2-A1:GFP plant (same section as in Fig. 3D). 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 p35S:COX4:GFP 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 pSEOR2:SEOR2:GFP 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.

Mentions: The subcellular organization of sieve elements was also examined. The GFP:LTI6b marker labeled the plasma membrane in the sieve elements (1–3 μm diameter in minor veins and 3–4 μm in larger veins), with brighter fluorescence was observed in the vicinity of the sieve plates (Fig. 3 B). Plants expressing the ER marker (ER:YFP) displayed a succession of fine fluorescent stacks at lateral positions in sieve elements (Fig. 4 A). For the mitochondrial marker (COX4:YFP), we observed faint fluorescence colocalized with the vital dye MitoTracker Red (Fig. 4 B,C), indicating this marker is active in sieve elements. Other fluorescent GFP markers were more difficult to visualize. For the nuclear marker (H2B:RFP) or the tonoplast marker (γTIP:YFP), the breakdown of the nucleus and tonoplast during differentiation of the sieve elements accounted for the absence of fluorescence. For the actin marker (fABD2:GFP), the microtubule marker (GFP:MBD) and the chloroplast marker (RbcS:YFP), no fluorescence signal was detected in the sieve elements. Sieve element plastids are not photosynthetic, and RuBisCO is probably degraded during sieve element differentiation. The actin cytoskeleton has been identified in sieve elements [14, 26], and the lack of fluorescence may be due to the signal being below the detection threshold, or the GFP and YFP fusions being unable to pass through the PPUs between the companion cells and sieve elements.


Live imaging of companion cells and sieve elements in Arabidopsis leaves.

Cayla T, Batailler B, Le Hir R, Revers F, Anstead JA, Thompson GA, Grandjean O, Dinant S - PLoS ONE (2015)

Imaging of subcellular compartments and known protein bodies in sieve elements.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 p35S:ER:YFP x pSUC2:PP2-A1:GFP plant (same section as in Fig. 3D). 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 p35S:COX4:GFP 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 pSEOR2:SEOR2:GFP 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4340910&req=5

pone.0118122.g004: Imaging of subcellular compartments and known protein bodies in sieve elements.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 p35S:ER:YFP x pSUC2:PP2-A1:GFP plant (same section as in Fig. 3D). 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 p35S:COX4:GFP 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 pSEOR2:SEOR2:GFP 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.
Mentions: The subcellular organization of sieve elements was also examined. The GFP:LTI6b marker labeled the plasma membrane in the sieve elements (1–3 μm diameter in minor veins and 3–4 μm in larger veins), with brighter fluorescence was observed in the vicinity of the sieve plates (Fig. 3 B). Plants expressing the ER marker (ER:YFP) displayed a succession of fine fluorescent stacks at lateral positions in sieve elements (Fig. 4 A). For the mitochondrial marker (COX4:YFP), we observed faint fluorescence colocalized with the vital dye MitoTracker Red (Fig. 4 B,C), indicating this marker is active in sieve elements. Other fluorescent GFP markers were more difficult to visualize. For the nuclear marker (H2B:RFP) or the tonoplast marker (γTIP:YFP), the breakdown of the nucleus and tonoplast during differentiation of the sieve elements accounted for the absence of fluorescence. For the actin marker (fABD2:GFP), the microtubule marker (GFP:MBD) and the chloroplast marker (RbcS:YFP), no fluorescence signal was detected in the sieve elements. Sieve element plastids are not photosynthetic, and RuBisCO is probably degraded during sieve element differentiation. The actin cytoskeleton has been identified in sieve elements [14, 26], and the lack of fluorescence may be due to the signal being below the detection threshold, or the GFP and YFP fusions being unable to pass through the PPUs between the companion cells and sieve elements.

Bottom Line: The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix.GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids.The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institut Jean-Pierre Bourgin, INRA-AgroParisTech, UMR1318, ERL CNRS 3559, Saclay Plant Sciences, Versailles, France.

ABSTRACT
The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

Show MeSH