Ca2+ rise and nitric oxide (NO) generation are essential early steps in plant innate immunity and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this laboratory has demonstrated that a loss-of-function mutation of an Arabidopsis (Arabidopsis thaliana) plasma membrane Ca 2+ -permeable inwardly conducting ion channel impairs HR and that this phenotype could be rescued by the application of a NO donor. At present, the mechanism linking cytosolic Ca 2+ rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca 2+ / calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca 2+ signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild-type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and the HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca 2+ elevation, excluding the possibility of CaM acting upstream from Ca 2+ . The CaM antagonist and Ca 2+ chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca 2+ /CaM dependent in vitro. The CaM-like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and [avirulent] pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogenassociated molecular pattern-mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca 2+ influx into the cytosol activates CaM and/or a CML, which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade, leading to innate immune responses, including the HR.

Ca 2+ influx is an early signal initiating cytosolic immune responses to pathogen perception in plant cells; molecular components linking pathogen recognition to Ca 2+ influx are not delineated. Work presented here provides insights into this biological system of non-self recognition and response activation. We have recently identified a cyclic nucleotide-activated ion channel as facilitating the Ca 2+ flux that initiates immune signaling in the plant cell cytosol. Work in this report shows that elevation of cAMP is a key player in this signaling cascade. We show that cytosolic Ca 2+ elevation, nitric oxide (NO) and reactive oxygen species generation, as well as immune signaling, lead to a hypersensitive response upon application of pathogens and/or conserved molecules that are components of microbes and are all dependent on cAMP generation. Exogenous cAMP leads to Ca 2+ channel-dependent cytosolic Ca 2+ elevation, NO generation, and defense response gene expression in the absence of the non-self pathogen signal. Inoculation of leaves with a bacterial pathogen leads to cAMP elevation coordinated with Ca 2+ rise. cAMP acts as a secondary messenger in plants; however, no specific protein has been heretofore identified as activated by cAMP in a manner associated with a signaling cascade in plants, as we report here. Our linkage of cAMP elevation in pathogen-inoculated plant leaves to Ca 2+ channels and immune signaling downstream from cytosolic Ca 2+ elevation provides a model for how non-self detection can be transduced to initiate the cascade of events in the cell cytosol that orchestrate pathogen defense responses.

Ca 2+ and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca 2+ . The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca 2+ modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of a NO donor. At present, the interrelationship between Ca 2+ and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca 2+ uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca 2+ accumulation is reduced in dnd1 leaves compared to the wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence-associated genes, H 2 O 2 generation, lipid peroxidation, tissue necrosis, and increased salicylic acid levels) were more prominent in dnd1 leaves compared to the wild type. Application of a Ca 2+ channel blocker hastened senescence of detached wild-type leaves maintained in the dark, increasing the rate of chlorophyll loss, expression of a senescence-associated gene, and lipid peroxidation. Pharmacological manipulation of Ca 2+ signaling provides evidence consistent with genetic studies of the relationship between Ca 2+ signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild-type plants. Application of a NO donor effectively rescues many dnd1 senescence-related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca 2+ uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream basal NO production in addition to its role (also linked to NO signaling) in pathogen defense responses and that this NO generation acts as a negative regulator during plant leaf senescence signaling.