Measurement of cellular cGMP in plant cells and tissues using the endogenous fluorescent reporter FlincG

P>The cyclic nucleotide cGMP has been shown to play important roles in plant development and responses to abiotic and biotic stress. To date, the techniques that are available to measure cGMP in plants are limited by low spatial and temporal resolution. In addition, tissue destruction is necessary. To circumvent these drawbacks we have used the delta-FlincG fluorescent protein to create an endogenous cGMP sensor that can report cellular cGMP levels with high resolution in time and space in living plant cells. delta-FlincG in transient and stably expressing cells shows a dissociation constant for cGMP of around 200 nm giving it a dynamic range of around 20-2000 nm. Stimuli that were previously shown to alter cGMP in plant cells (nitric oxide and gibberrellic acid) evoked pronounced fluorescence signals in single cells and in root tissues, providing evidence that delta-FlincG reports changes in cellular cGMP in a physiologically relevant context.