It was recently suggested that the protein Cand2 acts as a G protein–coupled receptor (GPCR) for melatonin in Arabidopsis by mediating stomatal closure via H2O2 production and Ca2+ influx. Here, we examined whether Cand2 is indeed a melatonin receptor. Contrary to previous reports, confocal microscopy analyses indicated that Cand2 protein is localized in the cytoplasm rather than the plasma membrane. The role of Cand2 was further investigated in genetic analyses using two Arabidopsis cand2 knockout mutant lines, SALK_071302 (cand2-1) and SALK_068848 (cand2-2). We found that melatonin-mediated mitogen-activated protein kinase (MAPK) activation was not abolished in the cand2 mutant lines, nor did melatonin-mediated defense gene induction (e.g., GST1) change relative to that in the wild type Col-0. Following ER stress, the two cand2 mutant lines were identical to Col-0 in terms of defense gene induction, ion leakage, and ROS levels. Two G protein mutants, gpa1 (Gα mutant) and agb1 (Gβ mutant), also exhibited no disturbance in melatonin-mediated defense gene induction or melatonin-mediated MAPK activation. Collectively, our data indicate that Cand2 is neither a phytomelatonin receptor localized in the plasma membrane nor is it involved in the melatonin-mediated defense signaling pathway via G protein components. However, it remains unclear how melatonin-mediated MAPK activation was slightly decreased in the mutant cand2-2 without affecting downstream defense gene induction. Also, it cannot rule out the possibility that Cand2 may be a melatonin binding protein and that its binding may result in a decrease of free melatonin level in plants.
Vertebrates originally possess four functional subtypes of G protein-coupled melatonin receptor
