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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 Ca2+ elevations induced by high salt stimulus applied to roots. (A) Free Ca2+ elevations on each leaf were analyzed with ImageJ. Velocities (mm/s) of Ca2+ responses were determined for each leaf along paths figured by red dashed arrows (values of Ca2+ response velocity are presented on Table 1). (B) Propagation speeds along the main leaf vain are indicated for selected points (same space scale as in A). False color scale is in mm/s.
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Figure 7: Analysis of the propagation of Ca2+ elevations induced by high salt stimulus applied to roots. (A) Free Ca2+ elevations on each leaf were analyzed with ImageJ. Velocities (mm/s) of Ca2+ responses were determined for each leaf along paths figured by red dashed arrows (values of Ca2+ response velocity are presented on Table 1). (B) Propagation speeds along the main leaf vain are indicated for selected points (same space scale as in A). False color scale is in mm/s.

Mentions: Light emitted by G5A in intact plants facing a salt stress therefore appeared to be sufficiently intense to image the propagation of Ca2+ signals in leaves with good time resolution. We performed a simple analysis of Ca2+ waves on each leaf of plants subjected to a salt stress applied to roots. Ca2+ signal velocities were then calculated for each leaf of the plant shown in Figure 6 (red dashed arrows, Figure 7). This shows that velocity was not constant within a given leaf (it decreased at leaf tip) and differed depending on the leaf. Detailed numerical values are given (Table 1): maximum and minimum of velocities were 0.52 and 0.03 mm/s respectively.


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 Ca2+ elevations induced by high salt stimulus applied to roots. (A) Free Ca2+ elevations on each leaf were analyzed with ImageJ. Velocities (mm/s) of Ca2+ responses were determined for each leaf along paths figured by red dashed arrows (values of Ca2+ response velocity are presented on Table 1). (B) Propagation speeds along the main leaf vain 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 7: Analysis of the propagation of Ca2+ elevations induced by high salt stimulus applied to roots. (A) Free Ca2+ elevations on each leaf were analyzed with ImageJ. Velocities (mm/s) of Ca2+ responses were determined for each leaf along paths figured by red dashed arrows (values of Ca2+ response velocity are presented on Table 1). (B) Propagation speeds along the main leaf vain are indicated for selected points (same space scale as in A). False color scale is in mm/s.
Mentions: Light emitted by G5A in intact plants facing a salt stress therefore appeared to be sufficiently intense to image the propagation of Ca2+ signals in leaves with good time resolution. We performed a simple analysis of Ca2+ waves on each leaf of plants subjected to a salt stress applied to roots. Ca2+ signal velocities were then calculated for each leaf of the plant shown in Figure 6 (red dashed arrows, Figure 7). This shows that velocity was not constant within a given leaf (it decreased at leaf tip) and differed depending on the leaf. Detailed numerical values are given (Table 1): maximum and minimum of velocities were 0.52 and 0.03 mm/s respectively.

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