ABSTRACT: Cyclic adenosine monophosphate (3’,5’-cAMP) is a well-characterized and evolutionary conserved second messenger. In contrast to the animal cells, however, the role of 3’,5’-cAMP in plants remains enigmatic. With the exception of cyclic nucleotide-gated ion channels, no plant 3’,5’-cAMP interactors have been reported to date. To address the existing gap, we resorted to thermal proteome profiling (TPP) as novel biochemical approach for the identification of protein binders of a small-molecule of choice. Based on the significant change in the thermal stability caused by 3’,5’-cAMP, we report a list of 51 putative 3’,5’-cAMP protein interactors, of which we focused on actin isovatriant ACT2. Most importantly we could demonstrate that 3’,5’-cAMP binding increases the rate of actin polymerization, resulting in longer actin filaments. Moreover, 3’,5’-cAMP treatment affected actin bundling, resulting in thicker actin bundles, as observed in the Arabidopsis hypocotyl cells. In line, with the obtained results 3’,5’-cAMP supplementation resulted in partial rescue of the short hypocotyl phenotype of the act2act7 Arabidopsis mutant, and 3’,5’-cAMP treatment suppressed actin depolymerization induced by Latrunculin B. Finally, 3’,5’-cAMP effects were independent from its degradation to adenosine, and true for nanomolar concentrations of 3’,5’-cAMP reported for the plant cells. Considering evolutionary conservation of 3’,5’-cAMP and actin we expect that our findings will be relevant to other eukaryotic organisms, and as such demonstrate a novel and direct mechanism of 3’,5’-cAMP regulation of the actin cytoskeleton dynamics.
Marcin Luzarowski 