Nitric Oxide in Plants: Production and Cross-talk with Ca2+ Signaling Besson-Bard Angélique , Courtois Cécile , Gauthier Adrien , Dahan Jennifer , Dobrowolska Grazyna , Jeandroz Sylvain , Pugin Alain , Wendehenne David   Molecular Plant  Volume 1, Issue 2, Pages 218-228 (March 2008) DOI: 10.1093/mp/ssm016 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Cryptogein-Induced Early Events in Ammonium-Grown Tobacco Cell Suspensions. (A) Time course of nitrate efflux induced by cryptogein (25 nM) in ammonium-grown Nicotiana tabacum cv Xanthi cells deprived of nitrate (cry NH4+) and in nitrate-grown Nicotiana tabacum cv Xanthi cells (cry NO3−). Nitrate content was determined as described by Wendehenne et al. (2002). Each value represents the mean ± SD of nine measurements (three replicates per experiment performed three times). (B) NO production induced by cryptogein (25 nM) in ammonium-grown Nicotiana tabacum cv Xanthi cells deprived of nitrate (cry NH4+) and in nitrate-grown Nicotiana tabacum cv Xanthi cells (cry NO3−). NO production was measured using the intracellular NO-sensitive fluorophore diaminofluoresceine-diacetate (DAF-2DA), as reported by Lamotte et al. (2004). Each value represents the mean ± SD of nine measurements (three replicates per experiment performed three times). (C) Cryptogein-induced [Ca2+]cyt increase in ammonium-grown cells deprived of nitrate (cry NH4+) and in nitrate-grown cells (cry NO3−). [Ca2+]cyt measurement was performed using apoaequorin-transformed Nicotiana plumbaginifolia cells expressing aequorin addressed in the cytosol, as described by Lamotte et al. (2006). Controls, omitted for clarity, did not induce changes in [Ca2+]cyt during the time of experiment. Results are from one of three representative experiments. (D) H2O2 production induced by cryptogein (25 nM) in ammonium-grown Nicotiana tabacum cv Xanthi cells deprived of nitrate (cry NH4+) and in nitrate-grown Nicotiana tabacum cv Xanthi cells (cry NO3−). Production of H2O2 was detected by chemiluminescence, as reported by Lamotte et al. (2004). Each value represents the mean ± SD of nine measurements (three replicates per experiment performed three times). Molecular Plant 2008 1, 218-228DOI: (10.1093/mp/ssm016) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Nitric Oxide Induces an Elevation in [Ca2+]cyt in Nicotiana plumbaginifolia Cells. (A) Changes in [Ca2+]cyt in apoaequorin-transformed Nicotiana plumbaginifolia cells in response to DEA/NO. 8-br-cADPR (250 μM) was added to cell suspensions 10 min prior to DEA/NO (50 μM). Treatment with the DEA/NO solubilization buffer (control cells), omitted for clarity, induced the first increase of [Ca2+]cyt but not the second one, which was shown to be specific to NO (Lamotte et al., 2006). 8-br-cADPR itself, omitted for clarity, did not induce changes in [Ca2+]cyt during the time of experiment. Results are from one of six representative experiments. (B) Ineffectiveness of a second DEA/NO application in inducing changes of [Ca2+]cyt in apoaequorin-transformed Nicotiana plumbaginifolia cells. A second DEA/NO (50 μM) treatment was applied 30 min after the first treatment (time 0) with the NO donor (50 μM). Sorbitol (250 mM) was added to the cell suspensions 55 min after the beginning of the experiment. Results are from one of three representative experiments. Molecular Plant 2008 1, 218-228DOI: (10.1093/mp/ssm016) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 NO Promotes the Activation of the Protein Kinases NtOSAK and SIPK in Nicotiana plumbaginifolia Cells. (A) Activation of protein kinases in apoaequorin-transformed Nicotiana plumbaginifolia cells exposed to DEA/NO. Cell suspensions were treated with DEA/NO solubilization buffer (control) or 2 mM DEA/NO, and aliquots of the cultured cells were taken at the indicated times. The NO scavenger cPTIO (500 μM) was added to cell suspensions 10 min prior to DEA/NO, and aliquots were taken 30 min later. Protein kinase activities were analyzed in cell extracts by in-gel kinase assay with HIIIS as a substrate, as described by Lamotte et al. (2006). cPTIO itself did not induce protein kinase activation (data not shown). The 42-kDa protein kinase was identified as NtOSAK; its activation was induced in response to micromolar concentrations of DEA/NO (see the text; Lamotte et al., 2006). The results shown are one of five representative experiments. (B) Immunological analysis and Ca2+ dependence of the NO-activated 48-kDa protein kinase. Protein extracts from untreated apoaequorin-transformed Nicotiana plumbaginifolia cells or exposed to DEA/NO (2 mM) for 30 min were subjected to a Western blot analysis using polyclonal antibodies that are specific to the MAPK-activated form. Ruthenium red (RR, 5 μM) and La3+ (500 μM) were added to cell suspensions 10 min prior to DEA/NO. When used alone, these compounds did not induce MAPK activation (data not shown). The results shown are one of three representative experiments. (C) Effects of La3+ on the NO-induced activation of the 48-kDa protein kinase and NtOSAK in apoaequorin-transformed Nicotiana plumbaginifolia cells. La3+ (500 μM) was added to the cell suspensions 10 min prior to DEA/NO (2 mM). The cells were exposed to the donor for 15 min and then analyzed for protein kinase activity by in-gel kinase assay with HIIIS as a substrate. When used alone, La3+ did not induce protein kinase activation (data not shown). The results shown are one of three representative experiments. (D) Immunoprecipitation of the NO-activated 48-kDa protein kinase with the anti-SIPK antibodies. Protein extracts from untreated apoaequorin-transformed N. plumbaginifolia cells or exposed to DEA/NO (2 mM) for 30 min were immunoprecipitated with anti-SIPK antibodies. The resulting immunocomplexes were analyzed by in-gel kinase assay with HIIIS as a substrate. The results shown are one of three representative experiments. Molecular Plant 2008 1, 218-228DOI: (10.1093/mp/ssm016) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions