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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.


Construction and operating principle of the G5A Ca2+ reporter. (A) Structure of the chimeric gene encoding the G5A sensor. The open-reading frame (ORF) encoding the green fluorescent protein (GFP) is linked to the ORF encoding the apo-aequorin by five repeats of a short sequence encoding a SGGSGSGGQ oligopeptide. The structure and length of this linker ensures efficient bioluminescence resonance energy transfer (BRET) between the aequorin and the GFP (Baubet et al., 2000). Using the pGWB502Ω vector, constitutive G5A expression is driven by a 35S promoter. (B) Principle of the aequorin bioluminescence emission upon binding of Ca2+. In the presence of the coelenterazine cofactor (“C”), the apo-aequorin is reconstituted as a functional aequorin. Upon binding of three Ca2+ ions, the cofactor is released with emission of light at ~470 nm. (C) Principle of G5A fluorescence emission upon binding of Ca2+. Thanks to the tight molecular coupling between the aequorin and GFP moieties of G5A, a BRET phenomenon between aequorin and GFP leads to excitation of and fluorescence emission at 510 nm by the latter.
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Figure 1: Construction and operating principle of the G5A Ca2+ reporter. (A) Structure of the chimeric gene encoding the G5A sensor. The open-reading frame (ORF) encoding the green fluorescent protein (GFP) is linked to the ORF encoding the apo-aequorin by five repeats of a short sequence encoding a SGGSGSGGQ oligopeptide. The structure and length of this linker ensures efficient bioluminescence resonance energy transfer (BRET) between the aequorin and the GFP (Baubet et al., 2000). Using the pGWB502Ω vector, constitutive G5A expression is driven by a 35S promoter. (B) Principle of the aequorin bioluminescence emission upon binding of Ca2+. In the presence of the coelenterazine cofactor (“C”), the apo-aequorin is reconstituted as a functional aequorin. Upon binding of three Ca2+ ions, the cofactor is released with emission of light at ~470 nm. (C) Principle of G5A fluorescence emission upon binding of Ca2+. Thanks to the tight molecular coupling between the aequorin and GFP moieties of G5A, a BRET phenomenon between aequorin and GFP leads to excitation of and fluorescence emission at 510 nm by the latter.

Mentions: Calcium (Ca2+) has long been established as a second messenger. Transgenic expression of fluorescence resonance energy transfer (FRET)-based fluorescent Ca2+ reporters such as the popular yellow cameleon (YC) or of the bioluminescent aequorin has permitted non-invasive monitoring of free Ca2+ levels and enabled real-time imaging of Ca2+ levels in different cell-types and organisms, including plants (Knight et al., 1991; Perez Koldenkova and Nagai, 2013). The YC has been used extensively for imaging Ca2+ signals in specific plant cell types such as guard cells (Allen et al., 1999), germinating pollen tubes (Iwano et al., 2012), and root hairs (Miwa et al., 2006; Monshausen et al., 2008). YC is also well suitable for Ca2+ sensing in subcellular compartments (Krebs et al., 2012; Bonza et al., 2013). However, YC requires excitation by exogenous light, which limits its relevance in plant photosynthetic tissues due to high background emission from auto-fluorescent cell walls, chlorophyll, and secondary metabolites. Indeed, wide autofluorescent spectrum of plant leaf pigments that overlap YC emission limits visualization of changes in intensity of YC fluorescence emission upon Ca2+ elevation. Moreover, Ca2+ imaging at plant tissue level requires strong and long excitation to detect fluorescence signals. Long term Ca2+ measurements would result in some YC photo-bleaching and/or tissue damage, this limiting long term Ca2+ measurements, over 24 h for example. On the other hand, the bioluminescent Ca2+ reporter aequorin does not require exogenous excitation light and very little background signal is produced resulting in a high signal-to-noise ratio throughout long acquisition periods. Aequorin has the largest dynamic range among Ca2+ reporters, allowing the monitoring of Ca2+ signals over several days and over a wide range of Ca2+ concentrations (Alonso and Garcia-Sancho, 2011). Aequorin has been introduced into several plant species (Knight et al., 1991; Webb et al., 2010) and has enabled photon counting based monitoring of Ca2+ in intact plant leaves. Many reports of aequorin application in plants have been published, where photon counting with luminometers was used to describe Ca2+ signaling under several stress conditions. However, to image photons emitted by aequorin with good resolution in both space and time requires sophisticated detection devices such as image photon detectors (IPDs) (Webb et al., 2010) or cameras fitted with an Intensified Charge-Coupled Device (ICCD) (Webb et al., 2010) or Electron Multiplying Charge-Coupled Device (EMCCD) (Rogers et al., 2008; Webb et al., 2010). This is a significant limitation to in planta Ca2+ imaging which could be overcome by using the G5A probe, an engineered fusion between the green fluorescent protein (GFP) and aequorin (Figures 1A,C) initially developed for Ca2+ imaging in animal cells (Baubet et al., 2000; Rogers et al., 2005). Through a bioluminescence resonance energy transfer (BRET) from aequorin to GFP, the wavelength of the emitted photon is 510 nm, instead of 470 nm and detection yield by CCD is found optimized, compared to aequorin, with a better signal/noise ratio (Baubet et al., 2000; Rogers et al., 2005, 2008).


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)

Construction and operating principle of the G5A Ca2+ reporter. (A) Structure of the chimeric gene encoding the G5A sensor. The open-reading frame (ORF) encoding the green fluorescent protein (GFP) is linked to the ORF encoding the apo-aequorin by five repeats of a short sequence encoding a SGGSGSGGQ oligopeptide. The structure and length of this linker ensures efficient bioluminescence resonance energy transfer (BRET) between the aequorin and the GFP (Baubet et al., 2000). Using the pGWB502Ω vector, constitutive G5A expression is driven by a 35S promoter. (B) Principle of the aequorin bioluminescence emission upon binding of Ca2+. In the presence of the coelenterazine cofactor (“C”), the apo-aequorin is reconstituted as a functional aequorin. Upon binding of three Ca2+ ions, the cofactor is released with emission of light at ~470 nm. (C) Principle of G5A fluorescence emission upon binding of Ca2+. Thanks to the tight molecular coupling between the aequorin and GFP moieties of G5A, a BRET phenomenon between aequorin and GFP leads to excitation of and fluorescence emission at 510 nm by the latter.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Construction and operating principle of the G5A Ca2+ reporter. (A) Structure of the chimeric gene encoding the G5A sensor. The open-reading frame (ORF) encoding the green fluorescent protein (GFP) is linked to the ORF encoding the apo-aequorin by five repeats of a short sequence encoding a SGGSGSGGQ oligopeptide. The structure and length of this linker ensures efficient bioluminescence resonance energy transfer (BRET) between the aequorin and the GFP (Baubet et al., 2000). Using the pGWB502Ω vector, constitutive G5A expression is driven by a 35S promoter. (B) Principle of the aequorin bioluminescence emission upon binding of Ca2+. In the presence of the coelenterazine cofactor (“C”), the apo-aequorin is reconstituted as a functional aequorin. Upon binding of three Ca2+ ions, the cofactor is released with emission of light at ~470 nm. (C) Principle of G5A fluorescence emission upon binding of Ca2+. Thanks to the tight molecular coupling between the aequorin and GFP moieties of G5A, a BRET phenomenon between aequorin and GFP leads to excitation of and fluorescence emission at 510 nm by the latter.
Mentions: Calcium (Ca2+) has long been established as a second messenger. Transgenic expression of fluorescence resonance energy transfer (FRET)-based fluorescent Ca2+ reporters such as the popular yellow cameleon (YC) or of the bioluminescent aequorin has permitted non-invasive monitoring of free Ca2+ levels and enabled real-time imaging of Ca2+ levels in different cell-types and organisms, including plants (Knight et al., 1991; Perez Koldenkova and Nagai, 2013). The YC has been used extensively for imaging Ca2+ signals in specific plant cell types such as guard cells (Allen et al., 1999), germinating pollen tubes (Iwano et al., 2012), and root hairs (Miwa et al., 2006; Monshausen et al., 2008). YC is also well suitable for Ca2+ sensing in subcellular compartments (Krebs et al., 2012; Bonza et al., 2013). However, YC requires excitation by exogenous light, which limits its relevance in plant photosynthetic tissues due to high background emission from auto-fluorescent cell walls, chlorophyll, and secondary metabolites. Indeed, wide autofluorescent spectrum of plant leaf pigments that overlap YC emission limits visualization of changes in intensity of YC fluorescence emission upon Ca2+ elevation. Moreover, Ca2+ imaging at plant tissue level requires strong and long excitation to detect fluorescence signals. Long term Ca2+ measurements would result in some YC photo-bleaching and/or tissue damage, this limiting long term Ca2+ measurements, over 24 h for example. On the other hand, the bioluminescent Ca2+ reporter aequorin does not require exogenous excitation light and very little background signal is produced resulting in a high signal-to-noise ratio throughout long acquisition periods. Aequorin has the largest dynamic range among Ca2+ reporters, allowing the monitoring of Ca2+ signals over several days and over a wide range of Ca2+ concentrations (Alonso and Garcia-Sancho, 2011). Aequorin has been introduced into several plant species (Knight et al., 1991; Webb et al., 2010) and has enabled photon counting based monitoring of Ca2+ in intact plant leaves. Many reports of aequorin application in plants have been published, where photon counting with luminometers was used to describe Ca2+ signaling under several stress conditions. However, to image photons emitted by aequorin with good resolution in both space and time requires sophisticated detection devices such as image photon detectors (IPDs) (Webb et al., 2010) or cameras fitted with an Intensified Charge-Coupled Device (ICCD) (Webb et al., 2010) or Electron Multiplying Charge-Coupled Device (EMCCD) (Rogers et al., 2008; Webb et al., 2010). This is a significant limitation to in planta Ca2+ imaging which could be overcome by using the G5A probe, an engineered fusion between the green fluorescent protein (GFP) and aequorin (Figures 1A,C) initially developed for Ca2+ imaging in animal cells (Baubet et al., 2000; Rogers et al., 2005). Through a bioluminescence resonance energy transfer (BRET) from aequorin to GFP, the wavelength of the emitted photon is 510 nm, instead of 470 nm and detection yield by CCD is found optimized, compared to aequorin, with a better signal/noise ratio (Baubet et al., 2000; Rogers et al., 2005, 2008).

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.