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Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter.

Xiong TC, Ronzier E, Sanchez F, Corratgé-Faillie C, Mazars C, Thibaud JB - Front Plant Sci (2014)

Bottom Line: We describe a simple method to image Ca(2+) signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera.We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca(2+) signals.It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

View Article: PubMed Central - PubMed

Affiliation: Biochimie et Physiologie Moléculaire des Plantes, Institut National de la Recherche Agronomique, UMR 386 Montpellier, France ; Biochimie et Physiologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, UMR 5004 Montpellier, France ; Biochimie et Physiologie Moléculaire des Plantes SupAgro, Montpellier, France ; Biochimie et Physiologie Moléculaire des Plantes, UM2 Montpellier, France.

ABSTRACT
Calcium (Ca(2+)) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca(2+) signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca(2+) signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca(2+) signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca(2+) imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca(2+) sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca(2+)-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca(2+) signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca(2+) dynamics in intact plants. We describe a simple method to image Ca(2+) signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca(2+) signals. It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

No MeSH data available.


Related in: MedlinePlus

Analysis of the propagation of calcium elevation in detached leaves induced by high salt exposure. (A) Ca2+ responses on each vein were analyzed in ImageJ. Velocities (mm/s) of Ca2+ signals were determined along paths represented by dashed arrows (details of Ca2+ signal velocity, duration and latency are presented on Table 2; colors of dashed arrows refer to the latency values so that Ca2+ waves that started at similar times are in the same color). (B) Propagation speeds along the main leaf vein are indicated for selected points (same space scale as in A). False color scale is in mm/s.
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Figure 9: Analysis of the propagation of calcium elevation in detached leaves induced by high salt exposure. (A) Ca2+ responses on each vein were analyzed in ImageJ. Velocities (mm/s) of Ca2+ signals were determined along paths represented by dashed arrows (details of Ca2+ signal velocity, duration and latency are presented on Table 2; colors of dashed arrows refer to the latency values so that Ca2+ waves that started at similar times are in the same color). (B) Propagation speeds along the main leaf vein are indicated for selected points (same space scale as in A). False color scale is in mm/s.

Mentions: In this example (representative of five independent leaves), elevation of free Ca2+ induced by NaCl needed 315 s (image at 1:00 to 6:15) to travel through a 57 mm-long leaf. Thus, in this example the average velocity was 0.181 mm/s. A further analysis was performed on the primary vein of this leaf (Figure 8C, red dashed line X–Y), a kymographic representation of velocity value on the axis X–Y shows that there were three different Ca2+ response velocities (Figure 8D). Two of them (red dashed and orange dashed arrows) spread acropetally (from X to Y) whereas one propagated the opposite way (Y–X, cyan dashed arrow). Interestingly, between the first third and the second third of the leaf, was observed a region where no Ca2+ signals were detected with G5A. Despite this gap, Ca2+ signal propagation was observed all along the XY axis (Figure 8C and Supplementary video S3). Analyses of Ca2+ waves on this excised leaf (representative of five leaves) show that Ca2+ signal velocities along different veins (Figure 9A dashed arrows) were different. Local velocity values were plotted on the image (as spots in false-color scale, Figure 9B). Higher velocities in the center of the leaf and slower velocities at leaf borders were found (Figure 9B). Details of velocities of Ca2+ signals in leaf veins, including on X–Y axis (#1, #8, and #16) are presented in Table 2 below. Despite inevitable variation from a leaf excised from a plant to another leaf excised from another plant, the nature and pattern (both in space and time) was essentially reproducible (see Supplemental video S4).


Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter.

Xiong TC, Ronzier E, Sanchez F, Corratgé-Faillie C, Mazars C, Thibaud JB - Front Plant Sci (2014)

Analysis of the propagation of calcium elevation in detached leaves induced by high salt exposure. (A) Ca2+ responses on each vein were analyzed in ImageJ. Velocities (mm/s) of Ca2+ signals were determined along paths represented by dashed arrows (details of Ca2+ signal velocity, duration and latency are presented on Table 2; colors of dashed arrows refer to the latency values so that Ca2+ waves that started at similar times are in the same color). (B) Propagation speeds along the main leaf vein are indicated for selected points (same space scale as in A). False color scale is in mm/s.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927637&req=5

Figure 9: Analysis of the propagation of calcium elevation in detached leaves induced by high salt exposure. (A) Ca2+ responses on each vein were analyzed in ImageJ. Velocities (mm/s) of Ca2+ signals were determined along paths represented by dashed arrows (details of Ca2+ signal velocity, duration and latency are presented on Table 2; colors of dashed arrows refer to the latency values so that Ca2+ waves that started at similar times are in the same color). (B) Propagation speeds along the main leaf vein are indicated for selected points (same space scale as in A). False color scale is in mm/s.
Mentions: In this example (representative of five independent leaves), elevation of free Ca2+ induced by NaCl needed 315 s (image at 1:00 to 6:15) to travel through a 57 mm-long leaf. Thus, in this example the average velocity was 0.181 mm/s. A further analysis was performed on the primary vein of this leaf (Figure 8C, red dashed line X–Y), a kymographic representation of velocity value on the axis X–Y shows that there were three different Ca2+ response velocities (Figure 8D). Two of them (red dashed and orange dashed arrows) spread acropetally (from X to Y) whereas one propagated the opposite way (Y–X, cyan dashed arrow). Interestingly, between the first third and the second third of the leaf, was observed a region where no Ca2+ signals were detected with G5A. Despite this gap, Ca2+ signal propagation was observed all along the XY axis (Figure 8C and Supplementary video S3). Analyses of Ca2+ waves on this excised leaf (representative of five leaves) show that Ca2+ signal velocities along different veins (Figure 9A dashed arrows) were different. Local velocity values were plotted on the image (as spots in false-color scale, Figure 9B). Higher velocities in the center of the leaf and slower velocities at leaf borders were found (Figure 9B). Details of velocities of Ca2+ signals in leaf veins, including on X–Y axis (#1, #8, and #16) are presented in Table 2 below. Despite inevitable variation from a leaf excised from a plant to another leaf excised from another plant, the nature and pattern (both in space and time) was essentially reproducible (see Supplemental video S4).

Bottom Line: We describe a simple method to image Ca(2+) signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera.We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca(2+) signals.It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

View Article: PubMed Central - PubMed

Affiliation: Biochimie et Physiologie Moléculaire des Plantes, Institut National de la Recherche Agronomique, UMR 386 Montpellier, France ; Biochimie et Physiologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, UMR 5004 Montpellier, France ; Biochimie et Physiologie Moléculaire des Plantes SupAgro, Montpellier, France ; Biochimie et Physiologie Moléculaire des Plantes, UM2 Montpellier, France.

ABSTRACT
Calcium (Ca(2+)) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca(2+) signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca(2+) signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca(2+) signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca(2+) imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca(2+) sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca(2+)-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca(2+) signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca(2+) dynamics in intact plants. We describe a simple method to image Ca(2+) signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca(2+) signals. It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

No MeSH data available.


Related in: MedlinePlus