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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.


Ca2+ dynamics detected in response to dark onset from plants expressing G5A or aequorin. (A) Bright field view of batches of 7 day-old G5A (left) and Aeq (right) Arabidopsis reporter lines. Scale bar = 1 cm. (B) Cumulative amount of light emitted from these plants after dark onset, over 150 min, expressed in false colors (scale shown at right, in device-dependent arbitrary unit or “RLU,” standing for “relative light unit”). Representative results of 10 independent measurements of dark-induced Ca2+ released. (C) 150 min time course of the cumulative light emission over 60 s time lapses from plants in (A,B) (data are means ± SE of 10 independent experiments with four G5A and five Aeq plants). Ca2+ dynamics from the Aeq plants show kinetics (inset) similar to G5A plants but with approximately five times less light emission.
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Figure 5: Ca2+ dynamics detected in response to dark onset from plants expressing G5A or aequorin. (A) Bright field view of batches of 7 day-old G5A (left) and Aeq (right) Arabidopsis reporter lines. Scale bar = 1 cm. (B) Cumulative amount of light emitted from these plants after dark onset, over 150 min, expressed in false colors (scale shown at right, in device-dependent arbitrary unit or “RLU,” standing for “relative light unit”). Representative results of 10 independent measurements of dark-induced Ca2+ released. (C) 150 min time course of the cumulative light emission over 60 s time lapses from plants in (A,B) (data are means ± SE of 10 independent experiments with four G5A and five Aeq plants). Ca2+ dynamics from the Aeq plants show kinetics (inset) similar to G5A plants but with approximately five times less light emission.

Mentions: Sudden light-dark transition has been reported to induce weak Ca2+ signals in photosynthetic tissues (Johnson et al., 1995; Sai and Johnson, 2002; Dodd et al., 2006). To assess the capability of G5A to detect weak Ca2+ events in intact plant tissues, we challenged Arabidopsis plants with darkness: the reactions of G5A plants upon light-dark transition were compared to those of Aeq plants (Figure 5). Significantly more photons could be collected from G5A plants than from the Aeq plants over this period (Figure 5B). Successive integrations of photons over 1 min time lapses provided an overview of the Ca2+ signal kinetics (Figure 5C). Dark-induced Ca2+ signals displayed by G5A and aequorin had parallel kinetics (Figure 5C and inset), with a maximal light emission between 40 and 60 min. However, approximately five times more photons were detected from plants of the G5A line.


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)

Ca2+ dynamics detected in response to dark onset from plants expressing G5A or aequorin. (A) Bright field view of batches of 7 day-old G5A (left) and Aeq (right) Arabidopsis reporter lines. Scale bar = 1 cm. (B) Cumulative amount of light emitted from these plants after dark onset, over 150 min, expressed in false colors (scale shown at right, in device-dependent arbitrary unit or “RLU,” standing for “relative light unit”). Representative results of 10 independent measurements of dark-induced Ca2+ released. (C) 150 min time course of the cumulative light emission over 60 s time lapses from plants in (A,B) (data are means ± SE of 10 independent experiments with four G5A and five Aeq plants). Ca2+ dynamics from the Aeq plants show kinetics (inset) similar to G5A plants but with approximately five times less light emission.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Ca2+ dynamics detected in response to dark onset from plants expressing G5A or aequorin. (A) Bright field view of batches of 7 day-old G5A (left) and Aeq (right) Arabidopsis reporter lines. Scale bar = 1 cm. (B) Cumulative amount of light emitted from these plants after dark onset, over 150 min, expressed in false colors (scale shown at right, in device-dependent arbitrary unit or “RLU,” standing for “relative light unit”). Representative results of 10 independent measurements of dark-induced Ca2+ released. (C) 150 min time course of the cumulative light emission over 60 s time lapses from plants in (A,B) (data are means ± SE of 10 independent experiments with four G5A and five Aeq plants). Ca2+ dynamics from the Aeq plants show kinetics (inset) similar to G5A plants but with approximately five times less light emission.
Mentions: Sudden light-dark transition has been reported to induce weak Ca2+ signals in photosynthetic tissues (Johnson et al., 1995; Sai and Johnson, 2002; Dodd et al., 2006). To assess the capability of G5A to detect weak Ca2+ events in intact plant tissues, we challenged Arabidopsis plants with darkness: the reactions of G5A plants upon light-dark transition were compared to those of Aeq plants (Figure 5). Significantly more photons could be collected from G5A plants than from the Aeq plants over this period (Figure 5B). Successive integrations of photons over 1 min time lapses provided an overview of the Ca2+ signal kinetics (Figure 5C). Dark-induced Ca2+ signals displayed by G5A and aequorin had parallel kinetics (Figure 5C and inset), with a maximal light emission between 40 and 60 min. However, approximately five times more photons were detected from plants of the G5A line.

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.