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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 and new markers in sieve elements.Fluorescent proteins observed in leaves from plants carrying GFP expressed in different subcellular compartments. Images were obtained by CLSM. Fluorescence is shown in false color. (a) and (b) Observation of discrete bodies in a pRTM1:GFP:RTM1 plant. The bodies in the sieve elements are up to 1 μm in diameter. In (h) colocalization of GFP:RTM1 with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (c) and (d) Observation of fluorescence in a pRTM2:RTM2:GFP plant. The signal is mostly localized at the vicinity of the plasma membrane, within the sieve element. It is also observed around spherical bodies (*) located next to the plasma membrane. In (d) colocalization of RTM2:GFP with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (e)–(i) Observation of PP2-A1:GFP in a pSEOR2:PP2-A1:GFP plant. PP2-A1 fluorescence indicates the presence of PP2-A1 in discrete spots located at the borders of the cell and in the vicinity of the sieve plate. In (f) and (g), details of the localization of PP2-A1:GFP around organelles present in the sieve elements. Arrows indicates circular structures surrounded by PP2-A1. In (h) colocalization of PP2-A1:GFP with MitoTracker Red. GFP fluorescence is shown in green and MitoTracker fluorescence is shown in red. In (i) colocalization of PP2-A1:GFP with mitochondria. GFP fluorescence is shown in green, chloroplast autofluorescence in red and MitoTracker fluorescence is shown in blue. Arrows indicate mitochondria surrounded by PP2-A1:GFP. mi: mitochondrion. pl: chloroplast. sp: sieve plate. lpb: large protein body. spb: small protein body. Scale bar = 5 μm.
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pone.0118122.g005: Imaging of subcellular compartments and new markers in sieve elements.Fluorescent proteins observed in leaves from plants carrying GFP expressed in different subcellular compartments. Images were obtained by CLSM. Fluorescence is shown in false color. (a) and (b) Observation of discrete bodies in a pRTM1:GFP:RTM1 plant. The bodies in the sieve elements are up to 1 μm in diameter. In (h) colocalization of GFP:RTM1 with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (c) and (d) Observation of fluorescence in a pRTM2:RTM2:GFP plant. The signal is mostly localized at the vicinity of the plasma membrane, within the sieve element. It is also observed around spherical bodies (*) located next to the plasma membrane. In (d) colocalization of RTM2:GFP with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (e)–(i) Observation of PP2-A1:GFP in a pSEOR2:PP2-A1:GFP plant. PP2-A1 fluorescence indicates the presence of PP2-A1 in discrete spots located at the borders of the cell and in the vicinity of the sieve plate. In (f) and (g), details of the localization of PP2-A1:GFP around organelles present in the sieve elements. Arrows indicates circular structures surrounded by PP2-A1. In (h) colocalization of PP2-A1:GFP with MitoTracker Red. GFP fluorescence is shown in green and MitoTracker fluorescence is shown in red. In (i) colocalization of PP2-A1:GFP with mitochondria. GFP fluorescence is shown in green, chloroplast autofluorescence in red and MitoTracker fluorescence is shown in blue. Arrows indicate mitochondria surrounded by PP2-A1:GFP. mi: mitochondrion. pl: chloroplast. sp: sieve plate. lpb: large protein body. spb: small protein body. Scale bar = 5 μm.

Mentions: We analyzed the lines expressing the pRTM1:GFP:RTM1 and pRTM2:RTM2-GFP constructs in the TEV-susceptible C24 (rtm1/rtm1) and Col-O rtm2-1 mutant (rtm2/rtm2) background (Table 1), in which they provided functional complementation for TEV restriction with RTM1 [20, 27] and for LMV restriction with RMT2 (Revers, personal communication). In pRTM1:GFP:RTM1 plants, an alignment of spherical protein bodies with an apparent diameter of 1.16 ± 0.01 μm (n = 612) was found at the periphery of individual sieve elements (Fig. 5 A), with up to 30–40 bodies in the sieve elements found in the larger veins. These bodies were not colocalized with mitochondria (Fig. 5 B). Their dimensions were of a similar range to that of sieve element plastids [14, 28, 29]. By contrast in pRTM2:RTM2:GFP plants, a reticulate organization lining the plasma membrane and surrounding bodies smaller than mitochondria was observed in the sieve elements (Fig. 5 C,D). The nature of these bodies remains unknown, but structures of a similar dimension were observed in transmission electron micrographs of sieve elements in Arabidopsis leaves (S5 Fig.). In the lines expressing these sieve element fluorescent markers, as lines expressing companion fluorescent markers, similar findings were obtained for all orders of veins, other than for RTM2, which was difficult to image in the minor veins (Fig. 6).


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 and new markers in sieve elements.Fluorescent proteins observed in leaves from plants carrying GFP expressed in different subcellular compartments. Images were obtained by CLSM. Fluorescence is shown in false color. (a) and (b) Observation of discrete bodies in a pRTM1:GFP:RTM1 plant. The bodies in the sieve elements are up to 1 μm in diameter. In (h) colocalization of GFP:RTM1 with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (c) and (d) Observation of fluorescence in a pRTM2:RTM2:GFP plant. The signal is mostly localized at the vicinity of the plasma membrane, within the sieve element. It is also observed around spherical bodies (*) located next to the plasma membrane. In (d) colocalization of RTM2:GFP with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (e)–(i) Observation of PP2-A1:GFP in a pSEOR2:PP2-A1:GFP plant. PP2-A1 fluorescence indicates the presence of PP2-A1 in discrete spots located at the borders of the cell and in the vicinity of the sieve plate. In (f) and (g), details of the localization of PP2-A1:GFP around organelles present in the sieve elements. Arrows indicates circular structures surrounded by PP2-A1. In (h) colocalization of PP2-A1:GFP with MitoTracker Red. GFP fluorescence is shown in green and MitoTracker fluorescence is shown in red. In (i) colocalization of PP2-A1:GFP with mitochondria. GFP fluorescence is shown in green, chloroplast autofluorescence in red and MitoTracker fluorescence is shown in blue. Arrows indicate mitochondria surrounded by PP2-A1:GFP. mi: mitochondrion. pl: chloroplast. sp: sieve plate. lpb: large protein body. spb: small protein body. Scale bar = 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4340910&req=5

pone.0118122.g005: Imaging of subcellular compartments and new markers in sieve elements.Fluorescent proteins observed in leaves from plants carrying GFP expressed in different subcellular compartments. Images were obtained by CLSM. Fluorescence is shown in false color. (a) and (b) Observation of discrete bodies in a pRTM1:GFP:RTM1 plant. The bodies in the sieve elements are up to 1 μm in diameter. In (h) colocalization of GFP:RTM1 with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (c) and (d) Observation of fluorescence in a pRTM2:RTM2:GFP plant. The signal is mostly localized at the vicinity of the plasma membrane, within the sieve element. It is also observed around spherical bodies (*) located next to the plasma membrane. In (d) colocalization of RTM2:GFP with MitoTracker. GFP fluorescence is shown in green, plastid autofluorescence in red, and MitoTracker in blue. (e)–(i) Observation of PP2-A1:GFP in a pSEOR2:PP2-A1:GFP plant. PP2-A1 fluorescence indicates the presence of PP2-A1 in discrete spots located at the borders of the cell and in the vicinity of the sieve plate. In (f) and (g), details of the localization of PP2-A1:GFP around organelles present in the sieve elements. Arrows indicates circular structures surrounded by PP2-A1. In (h) colocalization of PP2-A1:GFP with MitoTracker Red. GFP fluorescence is shown in green and MitoTracker fluorescence is shown in red. In (i) colocalization of PP2-A1:GFP with mitochondria. GFP fluorescence is shown in green, chloroplast autofluorescence in red and MitoTracker fluorescence is shown in blue. Arrows indicate mitochondria surrounded by PP2-A1:GFP. mi: mitochondrion. pl: chloroplast. sp: sieve plate. lpb: large protein body. spb: small protein body. Scale bar = 5 μm.
Mentions: We analyzed the lines expressing the pRTM1:GFP:RTM1 and pRTM2:RTM2-GFP constructs in the TEV-susceptible C24 (rtm1/rtm1) and Col-O rtm2-1 mutant (rtm2/rtm2) background (Table 1), in which they provided functional complementation for TEV restriction with RTM1 [20, 27] and for LMV restriction with RMT2 (Revers, personal communication). In pRTM1:GFP:RTM1 plants, an alignment of spherical protein bodies with an apparent diameter of 1.16 ± 0.01 μm (n = 612) was found at the periphery of individual sieve elements (Fig. 5 A), with up to 30–40 bodies in the sieve elements found in the larger veins. These bodies were not colocalized with mitochondria (Fig. 5 B). Their dimensions were of a similar range to that of sieve element plastids [14, 28, 29]. By contrast in pRTM2:RTM2:GFP plants, a reticulate organization lining the plasma membrane and surrounding bodies smaller than mitochondria was observed in the sieve elements (Fig. 5 C,D). The nature of these bodies remains unknown, but structures of a similar dimension were observed in transmission electron micrographs of sieve elements in Arabidopsis leaves (S5 Fig.). In the lines expressing these sieve element fluorescent markers, as lines expressing companion fluorescent markers, similar findings were obtained for all orders of veins, other than for RTM2, which was difficult to image in the minor veins (Fig. 6).

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