title: Calcium-dependent regulation of genes for plant nodulation in Rhizobium leguminosarum detected by iTRAQ quantitative proteomic analysis.
abstract: Rhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant-microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca(2+) involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the nod gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca(2+) chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca(2+) influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed.
authors.affiliation: Proteomics Center of Padova University , Via G. Orus 2b, 35129 Padova, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Arrigoni
authors.forename: Serena
authors.initials: S
authors.lastname: Tolin
authors.forename: Roberto
authors.initials: R
authors.lastname: Moscatiello
authors.forename: Antonio
authors.initials: A
authors.lastname: Masi
authors.forename: Lorella
authors.initials: L
authors.lastname: Navazio
authors.forename: Andrea
authors.initials: A
authors.lastname: Squartini
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002853:Chromatography, Liquid; D004533:Egtazic Acid; D007887:Fabaceae / Q000737:chemistry / Q000382:microbiology*; D044950:Flavanones / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015394:Molecular Structure; D055170:Plant Root Nodulation / Q000235:genetics* / Q000502:physiology; D040901:Proteomics / Q000379:methods*; D016961:Rhizobium leguminosarum / Q000235:genetics* / Q000502:physiology; D013559:Symbiosis; D053719:Tandem Mass Spectrometry
chemical_list: D044950:Flavanones; D004533:Egtazic Acid; C005273:naringenin; D002118:Calcium