Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca2+ concentration ([Ca2+]cyt) in plants using Nicotiana plumbaginifolia cells expressing the Ca2+ reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca2+]cyt which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca2+]cyt changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca2+]cyt sensitive to plasma membrane Ca2+ channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca2+ channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca2+]cyt was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca2+]cyt by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.