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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 and free GFP in the phloem cells of a four-week-old leaf.Visualization of soluble GFP or PP2-A1 tagged with GFP or CFP in leaves from pSUC2:PP2-A1:GFP, pSUC2:PP2-A1:GFP, pSUC2:PP2-A1:CFP and pSUC2:GFP 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 SUC2 promoter, in a pSUC2:GFP 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 pSUC2:GFP 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 pSUC2:PP2-A1:GFP plant. Bent companion cells (indicated by *) are typically found at such junctions. (e) Localization of PP2-A1:GFP in a vein from a pSUC2:PP2-A1:GFP plant. GFP is found only in the companion cells. This observation was made on a cross between the pSUC2:PP2-A1:GFP (in green) and p35S:H2B:RFP 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 pSUC2:GFP:PP2-A1 plant. (g) Localization of PP2-A1:CFP fluorescence (shown in green) in a minor vein from a pSUC2:PP2-A1:CFP 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.
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pone.0118122.g002: Imaging of PP2-A1:GFP and free GFP in the phloem cells of a four-week-old leaf.Visualization of soluble GFP or PP2-A1 tagged with GFP or CFP in leaves from pSUC2:PP2-A1:GFP, pSUC2:PP2-A1:GFP, pSUC2:PP2-A1:CFP and pSUC2:GFP 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 SUC2 promoter, in a pSUC2:GFP 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 pSUC2:GFP 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 pSUC2:PP2-A1:GFP plant. Bent companion cells (indicated by *) are typically found at such junctions. (e) Localization of PP2-A1:GFP in a vein from a pSUC2:PP2-A1:GFP plant. GFP is found only in the companion cells. This observation was made on a cross between the pSUC2:PP2-A1:GFP (in green) and p35S:H2B:RFP 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 pSUC2:GFP:PP2-A1 plant. (g) Localization of PP2-A1:CFP fluorescence (shown in green) in a minor vein from a pSUC2:PP2-A1:CFP 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.

Mentions: Various transgenic Arabidopsis Col-O lines producing green fluorescent protein (GFP) or other fluorescent proteins (CFP or YFP) were generated for live imaging of the phloem (Table 1). Genes encoding free, soluble GFP or GFP fused to the phloem lectin PP2-A1 [19] were expressed under the control of a companion cell-specific promoter (Fig. 2 A, S1 Table). The SUC2 promoter [23] was used in all constructs in preference to the PP2-A1 promoter [19], which frequently drives cosuppression of the endogenous PP2-A1 gene (unpublished results). The veins were observed in vivo by confocal laser scanning microscopy (CLSM), immediately after leaf peeling. Fluorescence was bright in veins of the various orders (i.e. main, secondary and minor; Fig. 2 B). Longitudinal observations of the phloem cells were carried out. In pSUC2:PP2-A1:GFP plants, fluorescence was observed in the cytosol and nucleus of companion cells, with no fluorescence in the sieve elements or phloem parenchyma cells (PPCs) (Fig. 2 D,E). This subcellular distribution was identical to that in undetached leaves, as shown by in vivo observations of leaves in planta using stereo fluorescence microscope (S1 Fig.). Similar findings were recorded for all veins with the pSUC2:GFP:PP2-A1 and pSUC2:PP2-A1:CFP constructs (Fig. 2 F-G). In an individual confocal section, up to 20 companion cells could be observed in the main veins, whereas three to five companion cells were observed in the smallest minor veins. Similar images were obtained with the YFP-tagged PP2-A2, a phloem lectin closely related to PP2-A1 [19], in pSUC2:PP2-A2:YFP plants (S2 Fig., A). By contrast, free GFP fluorescence was observed in both phloem parenchyma cells and companion cells in pSUC2:GFP plants (Fig. 2 C). As the promoter sequence used was the same as that used for PP2 fusions, for which no signal was detected in phloem parenchyma cells, these findings demonstrate the high specificity of this promoter for companion cells, and they indicate that the soluble GFP was transported from the companion cell into the phloem parenchyma cells via the plasmodesmata. Phloem parenchyma cells were larger (up to 17 μm in diameter) and were found in lateral positions within the veins. By contrast, PP2 fusions, which have an apparent molecular weight (MWapp) of 55 and 45 kDa for the PP2-A1 and PP2-A2 fusions, respectively, did not diffuse from the companion cells to the phloem parenchyma cells, unlike free GFP (MWapp of 27 kDa), consistent with the plasmodesmata having a size exclusion limit for nonspecific trafficking of between 30 and 40 kDa in Arabidopsis leaves [24].


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 and free GFP in the phloem cells of a four-week-old leaf.Visualization of soluble GFP or PP2-A1 tagged with GFP or CFP in leaves from pSUC2:PP2-A1:GFP, pSUC2:PP2-A1:GFP, pSUC2:PP2-A1:CFP and pSUC2:GFP 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 SUC2 promoter, in a pSUC2:GFP 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 pSUC2:GFP 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 pSUC2:PP2-A1:GFP plant. Bent companion cells (indicated by *) are typically found at such junctions. (e) Localization of PP2-A1:GFP in a vein from a pSUC2:PP2-A1:GFP plant. GFP is found only in the companion cells. This observation was made on a cross between the pSUC2:PP2-A1:GFP (in green) and p35S:H2B:RFP 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 pSUC2:GFP:PP2-A1 plant. (g) Localization of PP2-A1:CFP fluorescence (shown in green) in a minor vein from a pSUC2:PP2-A1:CFP 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.
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pone.0118122.g002: Imaging of PP2-A1:GFP and free GFP in the phloem cells of a four-week-old leaf.Visualization of soluble GFP or PP2-A1 tagged with GFP or CFP in leaves from pSUC2:PP2-A1:GFP, pSUC2:PP2-A1:GFP, pSUC2:PP2-A1:CFP and pSUC2:GFP 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 SUC2 promoter, in a pSUC2:GFP 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 pSUC2:GFP 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 pSUC2:PP2-A1:GFP plant. Bent companion cells (indicated by *) are typically found at such junctions. (e) Localization of PP2-A1:GFP in a vein from a pSUC2:PP2-A1:GFP plant. GFP is found only in the companion cells. This observation was made on a cross between the pSUC2:PP2-A1:GFP (in green) and p35S:H2B:RFP 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 pSUC2:GFP:PP2-A1 plant. (g) Localization of PP2-A1:CFP fluorescence (shown in green) in a minor vein from a pSUC2:PP2-A1:CFP 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.
Mentions: Various transgenic Arabidopsis Col-O lines producing green fluorescent protein (GFP) or other fluorescent proteins (CFP or YFP) were generated for live imaging of the phloem (Table 1). Genes encoding free, soluble GFP or GFP fused to the phloem lectin PP2-A1 [19] were expressed under the control of a companion cell-specific promoter (Fig. 2 A, S1 Table). The SUC2 promoter [23] was used in all constructs in preference to the PP2-A1 promoter [19], which frequently drives cosuppression of the endogenous PP2-A1 gene (unpublished results). The veins were observed in vivo by confocal laser scanning microscopy (CLSM), immediately after leaf peeling. Fluorescence was bright in veins of the various orders (i.e. main, secondary and minor; Fig. 2 B). Longitudinal observations of the phloem cells were carried out. In pSUC2:PP2-A1:GFP plants, fluorescence was observed in the cytosol and nucleus of companion cells, with no fluorescence in the sieve elements or phloem parenchyma cells (PPCs) (Fig. 2 D,E). This subcellular distribution was identical to that in undetached leaves, as shown by in vivo observations of leaves in planta using stereo fluorescence microscope (S1 Fig.). Similar findings were recorded for all veins with the pSUC2:GFP:PP2-A1 and pSUC2:PP2-A1:CFP constructs (Fig. 2 F-G). In an individual confocal section, up to 20 companion cells could be observed in the main veins, whereas three to five companion cells were observed in the smallest minor veins. Similar images were obtained with the YFP-tagged PP2-A2, a phloem lectin closely related to PP2-A1 [19], in pSUC2:PP2-A2:YFP plants (S2 Fig., A). By contrast, free GFP fluorescence was observed in both phloem parenchyma cells and companion cells in pSUC2:GFP plants (Fig. 2 C). As the promoter sequence used was the same as that used for PP2 fusions, for which no signal was detected in phloem parenchyma cells, these findings demonstrate the high specificity of this promoter for companion cells, and they indicate that the soluble GFP was transported from the companion cell into the phloem parenchyma cells via the plasmodesmata. Phloem parenchyma cells were larger (up to 17 μm in diameter) and were found in lateral positions within the veins. By contrast, PP2 fusions, which have an apparent molecular weight (MWapp) of 55 and 45 kDa for the PP2-A1 and PP2-A2 fusions, respectively, did not diffuse from the companion cells to the phloem parenchyma cells, unlike free GFP (MWapp of 27 kDa), consistent with the plasmodesmata having a size exclusion limit for nonspecific trafficking of between 30 and 40 kDa in Arabidopsis leaves [24].

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
Related in: MedlinePlus