Moonlighting adenylyl cyclases in plants – anArabidopsis thaliana9-cis-epoxycarotenoid dioxygenase as point in case

Adenylyl cyclases (ACs) and their catalytic product cAMP are regulatory components of plant responses. AC domains are intrinsic components of complex molecules with multiple functions, some of which are co-regulated by cAMP. Here we used an amino acid search motif based on annotated ACs in organisms across species to identify 12 unique Arabidopsis thaliana candidate ACs, four of which have a role in the biosynthesis of the stress hormone abscisic acid (ABA). One of these, the 9- cis -epoxycarotenoid dioxygenase (NCED3, At3g14440), was identified by sequence and structural analysis as a putative AC and then tested experimentally for activity. We show that an NCED3 AC fragment can complement an AC deficient E. coli mutant and this rescue is nullified when key amino acids in the AC motif are mutated. AC activity was also confirmed by tandem liquid chromatography mass spectrometry (LC-MS/MS). Our results are consistent with a moonlighting role for mononucleotide cyclases in multi-domain proteins that have at least one other distinct molecular function such as catalysis or ion channel activation and promise to yield new insights into tuning mechanisms of ABA dependent plant responses. Finally, our search method can also be applied to discover ACs in other species including Homo sapiens . Highlights An adenylyl cyclase (AC) catalytic center motif identifies novel ACs in plants ACs can moonlight in complex proteins with other enzymatic domains A 9- cis -epoxycarotenoid dioxygenase essential for abscisic acid synthesis contains an AC This finding implicates cAMP in abscisic acid synthesis and signaling