Armadillo repeat only proteins are required for the function of plant CNGC channels

Plant cells respond to a wide range of stimuli through intracellular calcium (Ca2+) signaling. Cyclic nucleotide-gated channels (CNGCs) are a major class of plant Ca2+ channels, with 20 homologs in Arabidopsis. These tetrameric plasma membrane proteins act downstream of diverse signals, such as phytohormones, extracellular damage, cell wall integrity or temperature. Here, we identify a class of plant-specific proteins, Armadillo Repeat Only (ARO), as essential regulators of possibly all plant CNGCs. Abrogation of functional sporophytic AROs results in a phenotypic pattern strongly reminiscent of CNGC dysfunction, including defects in root gravitropism, root hair growth and morphology, stomatal movement, and responses to extracellular ATP and the phytohormone auxin. aro2/3/4 mutants are fully resistant to the toxic effects caused by overexpression of CNGCs. AROs colocalize and physically interact with multiple CNGCs and modulate CNGC-dependent currents in Xenopus oocytes. Structural modeling and site-directed mutagenesis reveal AROs tetramer formation surrounding the CNGC channel, interacting via its IQ domain. Taken together, plant CNGC channels don’t act alone, but in a larger complex - channelosome, first of a kind in plants.