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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 in companion cells and phloem parenchyma cells.(a, c-h) Fluorescent proteins observed in leaves from plants resulting from crosses between pSUC2:PP2-A1:CFP 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 pSUC2:PP2-A1:CFP x p35S:RbcS:YFP 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 p35S:GFP:LTI6b 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 pSUC2:PP2-A1:CFP x p35S:H2B:RFP plant. Companion cells have square-like nuclei. (d) Observation of the endoplasmic reticulum in the companion cells of a pSUC2:PP2-A1:CFP x p35S:ER:YFP 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 pSUC2:PP2-A1:CFP x p35S:COX4:GFP 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 pSUC2:PP2-A1:CFP x p35S:yTIP:YFP plant. The image shows several vacuoles per companion cell. (g) Observation of actin network in the companion cells of a pSUC2:PP2-A1:CFP x p35S:FABD2:GFP 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 pSUC2:PP2-A1:CFP x p35S:GFP:MBD plant. Stars indicate the vacuoles. Scale bar = 5 μm.
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pone.0118122.g003: Imaging of subcellular compartments in companion cells and phloem parenchyma cells.(a, c-h) Fluorescent proteins observed in leaves from plants resulting from crosses between pSUC2:PP2-A1:CFP 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 pSUC2:PP2-A1:CFP x p35S:RbcS:YFP 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 p35S:GFP:LTI6b 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 pSUC2:PP2-A1:CFP x p35S:H2B:RFP plant. Companion cells have square-like nuclei. (d) Observation of the endoplasmic reticulum in the companion cells of a pSUC2:PP2-A1:CFP x p35S:ER:YFP 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 pSUC2:PP2-A1:CFP x p35S:COX4:GFP 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 pSUC2:PP2-A1:CFP x p35S:yTIP:YFP plant. The image shows several vacuoles per companion cell. (g) Observation of actin network in the companion cells of a pSUC2:PP2-A1:CFP x p35S:FABD2:GFP 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 pSUC2:PP2-A1:CFP x p35S:GFP:MBD plant. Stars indicate the vacuoles. Scale bar = 5 μm.

Mentions: Companion cells are highly compartmentalized (Fig. 2 G). Their subcellular organization was analyzed in crosses between the pSUC2:PP2-A1:CFP line and transgenic lines producing GFP or YFP fluorescent marker molecules (S2 Table) (Fig. 3, S4 Fig.). These cells could be identified on the basis of chloroplast alignment or CFP fluorescence from the PP2-A1:CFP fusion (Fig. 3 A). An example of this approach is provided by the analysis of a cross with the p35S:GFP:LTI6b line [25], which carries a plasma membrane marker. Fluorescence was readily observed at the plasma membrane (PM) in companion cells (Fig. 3 B). In p35S:H2B:RFP plants expressing the fluorescent histone 2B (H2B:RFP) nuclear marker, the nuclei of companion cells appeared dense and compacted (Fig. 3 C). Similarly, large numbers of mitochondria (Fig. 3 E; S2 Fig., B; S2 Movie) could be seen in the cytoplasm of the companion cells, along with the endoplasmic reticulum (Fig. 3 D). There were also numerous vacuoles (Fig. 3 F), with one vacuole often in contact with the nucleus, in addition to chloroplasts (Fig. 3 A). These results were obtained with plants expressing an endoplasmic reticulum marker (ER:YFP), the cytochrome c oxidase IV mitochondrial marker (COX4:YFP), the vacuolar aquaporin membrane marker (γTIP:YFP) and the RuBisCO marker (RbcS:YFP), respectively. We also observed the organization of the cytoskeleton in plants expressing an actin filament marker (fABD2:GFP) or a cortical microtubule marker (GFP:MBD) (Fig. 3 G-H). The organization of the actin filaments was difficult to determine with fABD2:GFP, because the fluorescence signal was weaker than for other markers (Fig. 3 G). A few thick bundles of actin filaments were observed at the cell periphery, mostly aligned along the long axis of the cell. Individual fine actin filaments were difficult to visualize. As observed in GFP:MBD plants, the cortical microtubules (CMTs) were aligned in companion cells, as in other cell types, in a transverse or oblique orientation, although heterogeneous arrays could be observed in some cells (Fig. 3 H, S2 Fig., C-E).


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 in companion cells and phloem parenchyma cells.(a, c-h) Fluorescent proteins observed in leaves from plants resulting from crosses between pSUC2:PP2-A1:CFP 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 pSUC2:PP2-A1:CFP x p35S:RbcS:YFP 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 p35S:GFP:LTI6b 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 pSUC2:PP2-A1:CFP x p35S:H2B:RFP plant. Companion cells have square-like nuclei. (d) Observation of the endoplasmic reticulum in the companion cells of a pSUC2:PP2-A1:CFP x p35S:ER:YFP 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 pSUC2:PP2-A1:CFP x p35S:COX4:GFP 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 pSUC2:PP2-A1:CFP x p35S:yTIP:YFP plant. The image shows several vacuoles per companion cell. (g) Observation of actin network in the companion cells of a pSUC2:PP2-A1:CFP x p35S:FABD2:GFP 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 pSUC2:PP2-A1:CFP x p35S:GFP:MBD plant. Stars indicate the vacuoles. Scale bar = 5 μm.
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Related In: Results  -  Collection

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Show All Figures
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pone.0118122.g003: Imaging of subcellular compartments in companion cells and phloem parenchyma cells.(a, c-h) Fluorescent proteins observed in leaves from plants resulting from crosses between pSUC2:PP2-A1:CFP 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 pSUC2:PP2-A1:CFP x p35S:RbcS:YFP 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 p35S:GFP:LTI6b 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 pSUC2:PP2-A1:CFP x p35S:H2B:RFP plant. Companion cells have square-like nuclei. (d) Observation of the endoplasmic reticulum in the companion cells of a pSUC2:PP2-A1:CFP x p35S:ER:YFP 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 pSUC2:PP2-A1:CFP x p35S:COX4:GFP 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 pSUC2:PP2-A1:CFP x p35S:yTIP:YFP plant. The image shows several vacuoles per companion cell. (g) Observation of actin network in the companion cells of a pSUC2:PP2-A1:CFP x p35S:FABD2:GFP 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 pSUC2:PP2-A1:CFP x p35S:GFP:MBD plant. Stars indicate the vacuoles. Scale bar = 5 μm.
Mentions: Companion cells are highly compartmentalized (Fig. 2 G). Their subcellular organization was analyzed in crosses between the pSUC2:PP2-A1:CFP line and transgenic lines producing GFP or YFP fluorescent marker molecules (S2 Table) (Fig. 3, S4 Fig.). These cells could be identified on the basis of chloroplast alignment or CFP fluorescence from the PP2-A1:CFP fusion (Fig. 3 A). An example of this approach is provided by the analysis of a cross with the p35S:GFP:LTI6b line [25], which carries a plasma membrane marker. Fluorescence was readily observed at the plasma membrane (PM) in companion cells (Fig. 3 B). In p35S:H2B:RFP plants expressing the fluorescent histone 2B (H2B:RFP) nuclear marker, the nuclei of companion cells appeared dense and compacted (Fig. 3 C). Similarly, large numbers of mitochondria (Fig. 3 E; S2 Fig., B; S2 Movie) could be seen in the cytoplasm of the companion cells, along with the endoplasmic reticulum (Fig. 3 D). There were also numerous vacuoles (Fig. 3 F), with one vacuole often in contact with the nucleus, in addition to chloroplasts (Fig. 3 A). These results were obtained with plants expressing an endoplasmic reticulum marker (ER:YFP), the cytochrome c oxidase IV mitochondrial marker (COX4:YFP), the vacuolar aquaporin membrane marker (γTIP:YFP) and the RuBisCO marker (RbcS:YFP), respectively. We also observed the organization of the cytoskeleton in plants expressing an actin filament marker (fABD2:GFP) or a cortical microtubule marker (GFP:MBD) (Fig. 3 G-H). The organization of the actin filaments was difficult to determine with fABD2:GFP, because the fluorescence signal was weaker than for other markers (Fig. 3 G). A few thick bundles of actin filaments were observed at the cell periphery, mostly aligned along the long axis of the cell. Individual fine actin filaments were difficult to visualize. As observed in GFP:MBD plants, the cortical microtubules (CMTs) were aligned in companion cells, as in other cell types, in a transverse or oblique orientation, although heterogeneous arrays could be observed in some cells (Fig. 3 H, S2 Fig., C-E).

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