| In plant Ca2+ ion acts as an important secondary messenger responding to a variety of environmental stimuli. The increase of cytoplasmic free Ca2+ ([ Ca2+]i) is involved in the signaling network triggered by some stimuli such as biotic and abiotic stress and phytohormones as well. It is possible that exogenous jasmonic acid (JA) can trigger the physiological and biochemical reactions in the cell by the increase of [ Ca2*]i. It has been reported that JA could induce the raising of [ Ca2+]i due to the release of Ca2+ from intracellular store rather than the influx of extracellular Ca2+ from apoplast. Another evidence, however, showed that JA has no effect on the [Ca2+], change.Although there is a relationship between JA stimulation and [Ca2+]i increase, very few evidence has been obtained with a direct measurement of [ Ca2+]i after JA treatment. In the present study, the [Ca2+]i was directly detected by means of fluorescence method in the leaf cell of 10-d -old seedlings in Arabidopsis. After the application of exogenous JA in different concentrations, the Ca2+ fluorescence in the leaf pre-loaded with Fluo-3/AM was measured by confocal laser scanning microscopy (CLSM). The results showed that fluorescence was observed within 1 min after JA treatment and reached a peak at 5 min. The optimun concentration of JA was 100 μmol/L.To evaluate the role of apoplast Ca2+ and calmodulin (CaM) in JA signaling, pharmacological effectors of Ca2+ influx across the plasma membrane, antagonist of IPs receptor and antagonist of CaM were used for the investigation. Moreover, the expression of JA response gene, JR1 and VSP, was also investigated. The main results were as follows:1) With the pretreatment of nifedipine, a nonpermeable L-type channel blocker, the fluorescence of [Ca2+]i induced by JA was inhibited in a dose dependent manner in the leaves and 250μmol/L nifedipine was most effective. Verapamil, another nonpermeable L-type channel blocker, had no significant effect on JA triggered increase of [Ca2+]i. Both basal level and JA-induced increase of [Ca2+]i were reduced upon pretreatment with heparin, an antagonist of IP3 receptor, and the level of [Ca2+]i only reachedthe level of the control which not treated with heparin and JA.2) After JA treatment, the JA response genes JR1 and VSP increased their expression markedly in 10d-old seedings. Nifedipine inhibited the JA-induced JR1 expression in a dose dependent manner, while verapamil had no effect on it. In the range of concentration used in the experiment, heparin and TMB-8, antother antagonist of IP3 receptor, had the positive effect on JA-triggered JR1 expression and the gene expression were up-regulated.3) W-7, an antagonist of CaM, inhibited the expression of JR1 and VSP, but W-5, a less potent analog of W-7, was found to be incapable of inhibiting the expression of JR1 and VSP under the same concentrations as W-7.4) The application of exogenous Ca2+ ion could mimic the JA partially to induce the gene expression of JR1 and support another evidence that apoplastic Ca2+ was involved in the JA response.The data reported here demonstrate that JA can trigger the increase of cytoplasmic free Ca2+ concentration. Apoplast Ca2+ and the intracellular Ca2+ store contributed to the rising of [Ca2*]j in JA signaling pathway. Nifedipine sensitive plasma membrane Ca2+ channel is a positive regulator of JA-induced [Ca2+]i increase and the gene expression of JR1, but the Ca2+ mobilized from IP3 sensitive calcium stores is a negative regulator of JR1 expression. Ca2+ exerts its functions through activate the CaM or CaM related proteins. Fine processes involved in modulation of the JA-triggered Ca2+ signaling network was discussed.
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