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A Nuclear-Targeted Cameleon Demonstrates Intranuclear Ca²⁺ Spiking in Medicago truncatula Root Hairs in Response to Rhizobial Nodulation Factors^1,[W],[OA]

Laboratory of Plant-Microbe Interactions, UMR CNRS-INRA 2594/441, F–31320 Castanet-Tolosan, France (B.J.S., M.C., A.C.T., J.F., D.G.B.); and Laboratory of Systems Analysis and Architecture (LAAS-CNRS), Complexe Scientifique de Rangueil, 31077 Toulouse, France (A.M.)

Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca²⁺ spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine whether NFs also elicit Ca²⁺ responses within the plant cell nucleus we have made use of a nucleoplasmin-tagged cameleon (NupYC2.1). Confocal microscopy using this nuclear-specific calcium reporter has revealed sustained and regular Ca²⁺ spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca²⁺ oscillations is blocked in M. truncatula nfp, dmi1, and dmi2 mutants, and unaltered in a dmi3 background, it is likely that intranuclear spiking lies on the established NF-dependent signal transduction pathway, leading to cytoplasmic calcium spiking. A semiautomated mathematical procedure has been developed to identify and analyze nuclear Ca²⁺ spiking profiles, and has revealed high cell-to-cell variability in terms of both periodicity and spike duration. Time-lapse imaging of the cameleon Förster resonance energy transfer-based ratio has allowed us to visualize the nuclear spiking variability in situ and to demonstrate the absence of spiking synchrony between adjacent growing root hairs. Finally, spatio-temporal analysis of the asymmetric nuclear spike suggests that the initial rapid increase in Ca²⁺ concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca²⁺ oscillations on both sides of the nuclear envelope raises major questions about the respective roles of the cytoplasmic and nuclear compartments in transducing this key endosymbiotic signal.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: David G. Barker (david.barker{at}toulouse.inra.fr).

^[W] The online version of this article contains Web-only data.

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www.plantphysiol.org/cgi/doi/10.1104/pp.109.142851

^* Corresponding author; e-mail david.barker{at}toulouse.inra.fr.

Received June 11, 2009; accepted August 11, 2009; published August 21, 2009.