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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.

Show MeSH
Organization of the phloem cells in mature Arabidopsis leaves.One straightforward criterion for distinguishing between phloem parenchyma cells (PPC), companion cells (CC) and sieve elements (SE) is the presence and positions of plastids (pl). In the CCs, plastids are aligned and occupy a large proportion of the cell volume. Numerous mitochondria (mi) are also found in each cell type. The nuclei (nu) and the vacuole (vac) have unusual shapes and positions in the CCs. Plastid size differs with cell type (1 μm in SE, 3 μm in CC and 4 μm in PPC), whereas mitochondrial size variations are more limited (0.6 μm in SE, 0.6 μm in CC and 0.7 μm in PPC).
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pone.0118122.g008: Organization of the phloem cells in mature Arabidopsis leaves.One straightforward criterion for distinguishing between phloem parenchyma cells (PPC), companion cells (CC) and sieve elements (SE) is the presence and positions of plastids (pl). In the CCs, plastids are aligned and occupy a large proportion of the cell volume. Numerous mitochondria (mi) are also found in each cell type. The nuclei (nu) and the vacuole (vac) have unusual shapes and positions in the CCs. Plastid size differs with cell type (1 μm in SE, 3 μm in CC and 4 μm in PPC), whereas mitochondrial size variations are more limited (0.6 μm in SE, 0.6 μm in CC and 0.7 μm in PPC).

Mentions: Fluorescent markers were used for the in vivo imaging of subcellular components of phloem cells in detached Arabidopsis leaves (Fig. 8). Observations were made for a few minutes, immediately after sample preparation. SE observations on detached leaves may be problematic due to hydraulic disruption, as the pressure is likely to drop, making it difficult to refill the sieve tubes, in leaves disconnected from the plant xylem [33]. We carried out an experiment with CFDA to check that the transport activity of the phloem was preserved for several minutes. We also confirmed that leaf peeling did not substantially modify the subcellular organization of sieve elements, with respect to other methods, in experiments using fluorescently tagged proteins or vital markers [14, 34]. Virtually identical methods have been used on intact leaves from Vicia faba and tobacco, for the visualization of subcellular structures in phloem cells [17, 34]. However, transport in the phloem is an integrative phenomenon operating at whole-plant level that is dependent on sources, sinks, a transport tube and a source of water; live imaging results for phloem cells over more extended periods should therefore be interpreted with caution.


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)

Organization of the phloem cells in mature Arabidopsis leaves.One straightforward criterion for distinguishing between phloem parenchyma cells (PPC), companion cells (CC) and sieve elements (SE) is the presence and positions of plastids (pl). In the CCs, plastids are aligned and occupy a large proportion of the cell volume. Numerous mitochondria (mi) are also found in each cell type. The nuclei (nu) and the vacuole (vac) have unusual shapes and positions in the CCs. Plastid size differs with cell type (1 μm in SE, 3 μm in CC and 4 μm in PPC), whereas mitochondrial size variations are more limited (0.6 μm in SE, 0.6 μm in CC and 0.7 μm in PPC).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118122.g008: Organization of the phloem cells in mature Arabidopsis leaves.One straightforward criterion for distinguishing between phloem parenchyma cells (PPC), companion cells (CC) and sieve elements (SE) is the presence and positions of plastids (pl). In the CCs, plastids are aligned and occupy a large proportion of the cell volume. Numerous mitochondria (mi) are also found in each cell type. The nuclei (nu) and the vacuole (vac) have unusual shapes and positions in the CCs. Plastid size differs with cell type (1 μm in SE, 3 μm in CC and 4 μm in PPC), whereas mitochondrial size variations are more limited (0.6 μm in SE, 0.6 μm in CC and 0.7 μm in PPC).
Mentions: Fluorescent markers were used for the in vivo imaging of subcellular components of phloem cells in detached Arabidopsis leaves (Fig. 8). Observations were made for a few minutes, immediately after sample preparation. SE observations on detached leaves may be problematic due to hydraulic disruption, as the pressure is likely to drop, making it difficult to refill the sieve tubes, in leaves disconnected from the plant xylem [33]. We carried out an experiment with CFDA to check that the transport activity of the phloem was preserved for several minutes. We also confirmed that leaf peeling did not substantially modify the subcellular organization of sieve elements, with respect to other methods, in experiments using fluorescently tagged proteins or vital markers [14, 34]. Virtually identical methods have been used on intact leaves from Vicia faba and tobacco, for the visualization of subcellular structures in phloem cells [17, 34]. However, transport in the phloem is an integrative phenomenon operating at whole-plant level that is dependent on sources, sinks, a transport tube and a source of water; live imaging results for phloem cells over more extended periods should therefore be interpreted with caution.

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