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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 PP2-A1:GFP in sieve elements undergoing differentiation.Fluorescent PP2-A1:GFP protein observed in leaves of pSEOR2:PP2-A1:GFP 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.
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pone.0118122.g007: Imaging of PP2-A1:GFP in sieve elements undergoing differentiation.Fluorescent PP2-A1:GFP protein observed in leaves of pSEOR2:PP2-A1:GFP 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.

Mentions: The SEOR2 promoter is active in immature sieve elements [32], making it possible to localize PP2-A1:GFP in differentiating sieve elements. In pSEOR2:PP2-A1:GFP plants, differentiating sieve elements were readily recognizable on the basis of their bright fluorescence signal. Differentiation could be monitored by staining nuclei with propidium iodide and recording plastid autofluorescence. At early stages of differentiation in cells that still contained a nucleus, PP2-A1:GFP fluorescence was found in the cytoplasm and in the nucleus (Fig. 7 A), as in companion cells. In differentiating cells displaying plastid autofluorescence but with no observable nucleus, PP2-A1:GFP continued to accumulate in the cytosol (Fig. 7 B). At later stages, in cells retaining large organelles, presumably lytic vacuoles, but no longer displaying plastid autofluorescence and nuclear staining, PP2-A1:GFP was observed both in the cytosol and as small dots of fluorescence, indicating the progressive anchoring of PP2-A1 to mitochondria or other organelles (Fig. 7 C-E). In mature sieve elements, the cytosolic fluorescence had completely disappeared, and PP2-A1:GFP appeared as discrete spots (Fig. 7 F).


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 PP2-A1:GFP in sieve elements undergoing differentiation.Fluorescent PP2-A1:GFP protein observed in leaves of pSEOR2:PP2-A1:GFP 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118122.g007: Imaging of PP2-A1:GFP in sieve elements undergoing differentiation.Fluorescent PP2-A1:GFP protein observed in leaves of pSEOR2:PP2-A1:GFP 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.
Mentions: The SEOR2 promoter is active in immature sieve elements [32], making it possible to localize PP2-A1:GFP in differentiating sieve elements. In pSEOR2:PP2-A1:GFP plants, differentiating sieve elements were readily recognizable on the basis of their bright fluorescence signal. Differentiation could be monitored by staining nuclei with propidium iodide and recording plastid autofluorescence. At early stages of differentiation in cells that still contained a nucleus, PP2-A1:GFP fluorescence was found in the cytoplasm and in the nucleus (Fig. 7 A), as in companion cells. In differentiating cells displaying plastid autofluorescence but with no observable nucleus, PP2-A1:GFP continued to accumulate in the cytosol (Fig. 7 B). At later stages, in cells retaining large organelles, presumably lytic vacuoles, but no longer displaying plastid autofluorescence and nuclear staining, PP2-A1:GFP was observed both in the cytosol and as small dots of fluorescence, indicating the progressive anchoring of PP2-A1 to mitochondria or other organelles (Fig. 7 C-E). In mature sieve elements, the cytosolic fluorescence had completely disappeared, and PP2-A1:GFP appeared as discrete spots (Fig. 7 F).

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