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

Abstract

Ca 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 Ca and calmodulin (CaM). Here, we used transgenic seedlings expressing cyan fluorescent protein (CFP) containing a type 1 peroxisomal-targeting signal motif (PTS1; CFP-PTS1), which enables peroxisomes to be visualized, and also used a cell-permeable fluorescent probe for Ca Analysis by confocal laser-scanning microscopy (CLSM) enabled us to visualize the presence of endogenous Ca in the peroxisomes of both roots and guard cells. The presence of Ca 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 Ca 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.

Pharmacological Strategies for Manipulating Plant Ca2+ Signalling

“…Via such a vast array of potential targets, CaM antagonists elicit strong pleiotropic effects in plants. This ranges from secretion [ 280 ], mitotic progression [ 281 ], auxin transport [ 282 ], gravitropism [ 283 ], red and blue light-induced acidification by leaf epidermal cells [ 284 ], inhibition of cytokinin-induced bud formation in the moss Funaria [ 285 ], plant growth and defense [ 239 ], and peroxisomal Ca 2+ and protein import [ 286 , 287 ]. Additionally, several CaM inhibitors (W-7, TFP and calmidazolium) induce a cytosolic calcium increase [ 127 , 129 ] which itself could be the cause of some of the effects observed after CaM antagonist treatment, rather than a direct effect of CaM inhibition.…”

Plant peroxisomes at the crossroad of NO and H2O2 metabolism

“…The import of this NOS‐like protein into peroxisomes is dependent on peroxins PEX12 and PEX13 (Corpas et al ) and it seems to have a PTS type 2 (PTS2) (Corpas and Barroso ), like that of peroxisomal NOS from animal origins (Loughran et al ). More recently, it has been corroborated that the NO generation in peroxisomes is strictly dependent on calmodulin and calcium (Corpas and Barroso ). All these data have allowed us to get a more complete picture of the puzzle that represents the peroxisomal NO in plants.…”

Targeting and translocation of proteins to the endoplasmic reticulum at a glance

“…Additionally, Pex5 has been suggested to act in a PTS1-independent manner, which may explain the peroxisomal import of some proteins that lack a PTS (van der Klei and Veenhuis, 2006). Additional cytosolic factors are emerging as facilitators of protein import into peroxisomes, with the HSP40 Djp1 (Dobriyal et al, 2017;Hettema et al, 1998) and calmodulin (Corpas and Barroso, 2017) among them.…”