Vacuolar phosphatases are essential for efficient nucleotide salvage in Arabidopsis.

The salvage pathway that recycles nucleotides from RNA is an important contributor to cellular homeostasis. In Arabidopsis, RNA salvage occurs in the vacuole, in a process started by RNS2. Defects in this pathway lead to constitutive autophagy. How nucleosides are generated from RNS2-catalyzed RNA degradation remains unclear. Using a combination of biochemistry and molecular genetics, we showed that RNS2 produces 2',3'-cAMP and 3'-AMP from poly(A) degradation but only 2',3'-cUMP from poly(U). Mutants lacking PAP26, the major vacuolar acid phosphatase (APase), displayed increased basal autophagy that was rescued by inosine treatment, mirroring rns2 phenotypes. PAP26-deficient vacuoles have lower total APase activity than wild type, but nucleotide processing is not fully disrupted. Further analyses showed that VSP3 also contributes to the total vacuolar APase activity. Nucleotide metabolism in pap26 vsp3 double mutants is severely disrupted, and mutant vacuoles accumulate 3'-NMP, 5'-NMP, and, surprisingly, 2'-AMP. We propose that PAP26 and VSP3 are the main APases involved in vacuolar RNA salvage. In addition, our results suggest that other activities, including cyclic phosphodiesterases and possibly a 5'-NMP-producing exoribonuclease, are needed to facilitate this process in Arabidopsis, producing the metabolites that are transported to the cytoplasm to maintain nucleotide homeostasis.