Abstract
Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca(2+) spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine whether NFs also elicit Ca(2+) 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 Ca(2+) spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca(2+) 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 Ca(2+) 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 Ca(2+) concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca(2+) 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.
Highlights
Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca2+ spiking in the cytoplasmic perinuclear region of host legume root hairs
Since cytoplasmic Ca2+ spiking lies on the wellcharacterized NF transduction pathway that leads to the activation of host genes such as MtENOD11, we evaluated nuclear Ca2+ responses in mutant lines defective for each of the three Medicago genes (NFP, DMI1, and DMI2) that lie upstream of cytoplasmic Ca2+ spiking, as well as for the downstream DMI3 gene encoding the presumed calcium/calmodulin-dependent kinase (CCaMK) calcium decoder
Since its initial discovery over a decade ago by Ehrhardt et al (1996), NF-elicited cytoplasmic Ca2+ spiking in legume root hairs has been studied in considerable detail and integrated into a complex and still poorly understood signal transduction pathway leading from NF perception to specific gene expression
Summary
Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca2+ spiking in the cytoplasmic perinuclear region of host legume root hairs. To overcome the many limitations of microinjection, Miwa et al (2006) have reported the use of a Forster resonance energy transfer (FRET)based cameleon calcium sensor (YC2.1) to monitor cytoplasmic Ca2+ responses in transgenic M. truncatula roots These studies revealed that NFs elicit cellautonomous perinuclear Ca2+ spiking in root hairs and have provided evidence that the number of consecutive calcium spikes may be critical for regulating ENOD11 gene activation. Spatio-temporal image analysis has provided evidence that the steep initial increase in the nuclear Ca2+ level during spiking originates primarily at the nuclear periphery These findings raise fundamental questions about the mechanism and role of intracellular Ca2+ as a secondary messenger in the NF signal transduction pathway, and in particular the relationship between cytoplasmic and nuclear Ca2+ oscillatory responses
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