Calmodulin antagonist affects peroxisomal functionality by disrupting both peroxisomal Ca²⁺ and protein import

Ca²⁺ is a second messenger in many physiological and phytopathological processes. Peroxisomes are subcellular compartments with an active oxidative and nitrosative metabolism. Previous studies have demonstrated that peroxisomal nitric oxide (NO) generation is dependent on Ca²⁺ and calmodulin (CaM). Here, we used Arabidopsis thaliana transgenic seedlings expressing cyan fluorescent protein (CFP) containing a type 1 peroxisomal-targeting signal motif (PTS1; CFP-PTS1), which enables peroxisomes to be visualized in vivo, and also used a cell-permeable fluorescent probe for Ca²⁺ Analysis by confocal laser-scanning microscopy (CLSM) enabled us to visualize the presence of endogenous Ca²⁺ in the peroxisomes of both roots and guard cells. The presence of Ca²⁺ in peroxisomes and the import of CFP-PTS1 are drastically disrupted by both CaM antagonist and glutathione (GSH). Furthermore, the activity of three peroxisomal enzymes (catalase, glycolate oxidase and hydroxypyruvate reductase) containing PTS1 was clearly affected in these conditions, with a decrease of between 41 and 51%. In summary, data show that Ca²⁺ and CaM are strictly necessary for protein import and normal functionality of peroxisomal enzymes, including antioxidant and photorespiratory enzymes, as well as for NO production.