Dissection of nodulation signaling using pea mutants defective for calciumspiking induced by Nod factors and chitin oligomers

Changes in intracellular calcium in pea root hairs responding to Rhizobium leguminosarum by. viciae nodulation (Nod) factors were analyzed by using a microinjected calcium-sensitive fluorescent dye (dextran-linked Oregon Gree n). Within 1-2 min after Nod-factor addition, there was usually an increase in fluorescence, followed about 10 min later by spikes in fluorescence occ urring at a rate of about one spike per minute. These spikes, corresponding to an increase in calcium of approximate to 200 nM, were localized around the nuclear region, and they were similar in terms of lag and period to tho se induced by Nod factors in alfalfa. Calcium responses were analyzed in no nnodulating pea mutants, representing seven loci that affect early stages o f the symbiosis. Mutations affecting three loci (sym8, sym10, and sym19) ab olished Nod-factor-induced calcium spiking, whereas a normal response was s een in peas carrying alleles of sym2(A), sym7, sym9, and sym30. Chitin olig omers of four or five IV-acetylglucosamine residues could also induce calci um spiking, although the response was qualitatively different from that ind uced by Nod factors; a rapid increase in intracellular calcium was not obse rved, the period between spikes was lower, and the response was not as sust ained. The chitin-oligomer-induced calcium spiking did not occur in nodulat ion mutants (sym8, sym10, and sym19) that were defective for Nod-factor-ind uced spiking, suggesting that this response is related to nodulation signal ing. From our data and previous observations on the lack of mycorrhizal inf ection in some of the sym mutants, we propose a model for the potential ord er of pea nodulation genes in nodulation and mycorrhizal signaling.