- Paper report
Calcium dynamics in single plant cells
Genome Biology volume 1, Article number: reports024 (2000)
A new noninvasive technique has been developed that successfully measures calcium dynamics in single Arabidopsis guard cells.
Significance and context
This successful application of a new recombinant technique for measuring cytosolic calcium ([Ca2+]cyt), when combined with Arabidopsis genomics, will open up plant calcium signaling pathways to genetic analysis. The small size of most Arabidopsis cells makes microinjection of fluorescent calcium indicators into cells very difficult, and recombinant techniques that incorporate the gene for the luminescent calcium indicator aequorin into the genome have not been routinely successful at the level of the single cell. Allen et al. have used an ingenious recombinant construct made up of fluorescent proteins and the calcium-binding protein calmodulin - a 'cameleon' - as a calcium sensor in Arabidopsis guard cells. This first use of cameleons as calcium sensors in plants is an exciting and important advance that will enable researchers to tackle previously intractable questions in topics such as gravitropism, fertilization and cell differentiation at the level of the single plant cell.
In epidermal peels taken from plants transgenic for the calcium indicator yellow cameleon 2.1 (YC2.1), the cameleon protein was localized to the guard cell cytosol and no signal was reported from chloroplasts, suggesting that YC2.1 was not present in this organelle. The presence of YC2.1 in guard cells did not interfere with guard-cell closure of the stomatal pore induced by the plant signaling molecule abscisic acid (ABA). From the guard-cell response to extracellular calcium and ABA, the authors conclude that the calcium responses measured with YC2.1 compare well with those detected using microinjection of fluorescent calcium indicators. Although Allen et al. encountered some problems with chloroplast autofluorescence, they were able to investigate cytosolic calcium dynamics on a semi-quantitative basis. The cameleon technology enabled [Ca2+]cyt to be measured in both guard cells of a single stoma at the same time. This produced an unexpected result, clearly showing that stimulus-induced increases in [Ca2+]cyt were not synchronized in the two halves of the stomatal apparatus (although some stomata did show similar patterns). The authors also report cameleon localization in root tips and root hairs.
Cameleons are an ingenious application of fluorescence resonance energy transfer (FRET) to detect changes in calcium concentration. The cameleon is composed of two 'green fluorescent proteins' (GFPs) which are bound together via calmodulin and a calmodulin-binding peptide, M13, to form a Ca2+ sensor, as described in detail in Figure 1.
The authors identified seven separate advantages of using the cameleon technology over existing procedures. Probably the main advantage in Arabidopsis is the ability to study calcium homeostasis at the single-cell level. This will enable plant physiologists studying calcium signaling in plant cells to exploit the opportunities presented by advances in Arabidopsis genomics.
This first use of cameleons as calcium sensors in plants shows their potential for studying calcium homeostasis in individual cells. It is likely that the problems with chloroplast autofluorescence will be ironed out; however, even as it stands, the approach is a major advance. Although Allen et al. used isolated epidermal peels, it seems likely that with confocal microscopy the approach could be made totally noninvasive and applied to cells and tissues not previously amenable to experimentation. In addition, as the authors were careful to engineer multiple cloning sites upstream and downstream of the YC2.1 coding site in their expression vector, it will be possible to target YC2.1 to organelles and, through fusion to specific signaling proteins, to analyze highly localized calcium dynamics.
Allen CG, Kwak JM, Chu SP, Llopis J, Tsien RY, Harper JF, Schroeder JI: Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J. 1999, 19: 735-747. 0960-7412
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Carr, K., Hetherington, A. Calcium dynamics in single plant cells. Genome Biol 1, reports024 (2000). https://doi.org/10.1186/gb-2000-1-1-reports024