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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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Assay of CFDA transport in the phloem cells of a detached Arabidopsis leaf.Leaves were observed by epifluorescence microscopy, under a fluorescent stereo microscope. The fluorescence appears as false color presentation using ImageJ’ ‘FIRE’ LUT. (a, b) Observation of fluorescence after the application of CFDA to a treated area of the leaf. (a) The veins in the loading area are already fluorescently labeled after 8 s. (b) After 180 s, the labeling extends to the main vein, secondary vein and minor veins around the loading area. (c-f) Observation of the transport of the fluorescent label after the application of CFDA to the treated area. (c) Observation of leaf autofluorescence immediately after application of the tracer. Treated areas are less fluorescent, enhancing observation of the vascular network (white arrows). For this experiment, the superficial layers were peeled away from a single vein in two areas: the loading area (la) and the transport area (ta). CFDA was loaded in the loading area. (d) 10 seconds after the application of CFDA. (e) 40 seconds after the application of CFDA, the fluorescence is transported and is visible in the transport area. (f) 100 seconds after the application of CFDA, the fluorescence has moved beyond the transport area. La: loading area; ta: transport area; mv: minor vein, sv: secondary vein; v: main vein. Scale bar = 5 mm.
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pone.0118122.g001: Assay of CFDA transport in the phloem cells of a detached Arabidopsis leaf.Leaves were observed by epifluorescence microscopy, under a fluorescent stereo microscope. The fluorescence appears as false color presentation using ImageJ’ ‘FIRE’ LUT. (a, b) Observation of fluorescence after the application of CFDA to a treated area of the leaf. (a) The veins in the loading area are already fluorescently labeled after 8 s. (b) After 180 s, the labeling extends to the main vein, secondary vein and minor veins around the loading area. (c-f) Observation of the transport of the fluorescent label after the application of CFDA to the treated area. (c) Observation of leaf autofluorescence immediately after application of the tracer. Treated areas are less fluorescent, enhancing observation of the vascular network (white arrows). For this experiment, the superficial layers were peeled away from a single vein in two areas: the loading area (la) and the transport area (ta). CFDA was loaded in the loading area. (d) 10 seconds after the application of CFDA. (e) 40 seconds after the application of CFDA, the fluorescence is transported and is visible in the transport area. (f) 100 seconds after the application of CFDA, the fluorescence has moved beyond the transport area. La: loading area; ta: transport area; mv: minor vein, sv: secondary vein; v: main vein. Scale bar = 5 mm.

Mentions: We adapted the method described for Vicia faba [17], combining leaf peeling and light microscopy to view the vasculature of detached Arabidopsis leaves. This method yielded a higher resolution than could be obtained with untreated leaves. As sugar export capacity may decrease rapidly in Arabidopsis leaves following their excision from the plant [21], we investigated the possible impairment of phloem transport after the cutting of the petiole and peeling off of the leaf surface with a razor blade. We used the phloem symplasmic tracer 5,6 carboxyfluorescein-diacetate (CFDA) to investigate both phloem transport and sieve element integrity [22]. CFDA is a membrane-permeant dye that is cleaved by cellular esterase to release carboxyfluorescein (CF), a non membrane-permeant fluorescent form of the dye. Fluorescence rapidly progressed from the treated area into the veins (Fig. 1 A-B, S1 Movie), with CF reaching the main vein at an apparent velocity of 6–10 mm min-1, moving in a proximal direction toward the petiole of the detached leaf. This value was in the same range as the velocity determined in intact Arabidopsis plants (100 μm/s) [14], indicating that the treatment did not prevent phloem transport from the treated area to the petiole (i.e. sink-ward, as expected in intact leaves), and that leaf excision did not trigger the immediate sealing of the sieve tubes connected to the treated area. We also assessed the transport activity of the sieve tubes located immediately beneath the treated area. Two areas located 5 mm apart, close to the midrib, were peeled off, with one used for tracer application and the other, in a more proximal position, used for observations of tracer translocation (Fig. 1 C-F). After loading in the acropetal area, the tracer moved basipetally across the second area on its way to the main vein, at a flow velocity of up to 10 mm min-1 (Fig. 1 D-F). The fluorescence continued to progress along the veins in this observation area, with minimal fluorescence detectable outside the veins, indicating that the sieve elements were continuing to translocate material beneath the treated area. Thus, the treatment of abaxial tissues from a detached Arabidopsis leaf does not impede phloem transport function, at least during the first few minutes after cutting. Phloem imaging is, therefore, possible.


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)

Assay of CFDA transport in the phloem cells of a detached Arabidopsis leaf.Leaves were observed by epifluorescence microscopy, under a fluorescent stereo microscope. The fluorescence appears as false color presentation using ImageJ’ ‘FIRE’ LUT. (a, b) Observation of fluorescence after the application of CFDA to a treated area of the leaf. (a) The veins in the loading area are already fluorescently labeled after 8 s. (b) After 180 s, the labeling extends to the main vein, secondary vein and minor veins around the loading area. (c-f) Observation of the transport of the fluorescent label after the application of CFDA to the treated area. (c) Observation of leaf autofluorescence immediately after application of the tracer. Treated areas are less fluorescent, enhancing observation of the vascular network (white arrows). For this experiment, the superficial layers were peeled away from a single vein in two areas: the loading area (la) and the transport area (ta). CFDA was loaded in the loading area. (d) 10 seconds after the application of CFDA. (e) 40 seconds after the application of CFDA, the fluorescence is transported and is visible in the transport area. (f) 100 seconds after the application of CFDA, the fluorescence has moved beyond the transport area. La: loading area; ta: transport area; mv: minor vein, sv: secondary vein; v: main vein. Scale bar = 5 mm.
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pone.0118122.g001: Assay of CFDA transport in the phloem cells of a detached Arabidopsis leaf.Leaves were observed by epifluorescence microscopy, under a fluorescent stereo microscope. The fluorescence appears as false color presentation using ImageJ’ ‘FIRE’ LUT. (a, b) Observation of fluorescence after the application of CFDA to a treated area of the leaf. (a) The veins in the loading area are already fluorescently labeled after 8 s. (b) After 180 s, the labeling extends to the main vein, secondary vein and minor veins around the loading area. (c-f) Observation of the transport of the fluorescent label after the application of CFDA to the treated area. (c) Observation of leaf autofluorescence immediately after application of the tracer. Treated areas are less fluorescent, enhancing observation of the vascular network (white arrows). For this experiment, the superficial layers were peeled away from a single vein in two areas: the loading area (la) and the transport area (ta). CFDA was loaded in the loading area. (d) 10 seconds after the application of CFDA. (e) 40 seconds after the application of CFDA, the fluorescence is transported and is visible in the transport area. (f) 100 seconds after the application of CFDA, the fluorescence has moved beyond the transport area. La: loading area; ta: transport area; mv: minor vein, sv: secondary vein; v: main vein. Scale bar = 5 mm.
Mentions: We adapted the method described for Vicia faba [17], combining leaf peeling and light microscopy to view the vasculature of detached Arabidopsis leaves. This method yielded a higher resolution than could be obtained with untreated leaves. As sugar export capacity may decrease rapidly in Arabidopsis leaves following their excision from the plant [21], we investigated the possible impairment of phloem transport after the cutting of the petiole and peeling off of the leaf surface with a razor blade. We used the phloem symplasmic tracer 5,6 carboxyfluorescein-diacetate (CFDA) to investigate both phloem transport and sieve element integrity [22]. CFDA is a membrane-permeant dye that is cleaved by cellular esterase to release carboxyfluorescein (CF), a non membrane-permeant fluorescent form of the dye. Fluorescence rapidly progressed from the treated area into the veins (Fig. 1 A-B, S1 Movie), with CF reaching the main vein at an apparent velocity of 6–10 mm min-1, moving in a proximal direction toward the petiole of the detached leaf. This value was in the same range as the velocity determined in intact Arabidopsis plants (100 μm/s) [14], indicating that the treatment did not prevent phloem transport from the treated area to the petiole (i.e. sink-ward, as expected in intact leaves), and that leaf excision did not trigger the immediate sealing of the sieve tubes connected to the treated area. We also assessed the transport activity of the sieve tubes located immediately beneath the treated area. Two areas located 5 mm apart, close to the midrib, were peeled off, with one used for tracer application and the other, in a more proximal position, used for observations of tracer translocation (Fig. 1 C-F). After loading in the acropetal area, the tracer moved basipetally across the second area on its way to the main vein, at a flow velocity of up to 10 mm min-1 (Fig. 1 D-F). The fluorescence continued to progress along the veins in this observation area, with minimal fluorescence detectable outside the veins, indicating that the sieve elements were continuing to translocate material beneath the treated area. Thus, the treatment of abaxial tissues from a detached Arabidopsis leaf does not impede phloem transport function, at least during the first few minutes after cutting. Phloem imaging is, therefore, possible.

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