TOR mediates the autophagy response to altered nucleotide homeostasis in an RNase mutant

Abstract

The Arabidopsis thaliana T2 family endoribonuclease RNS2 localizes to the vacuole and functions in rRNA degradation. Loss of RNS2 activity impairs rRNA turnover and leads to constitutive autophagy, a process for degradation of cellular components. Autophagy is normally activated during environmental stress and is important for stress tolerance and homeostasis. Here we show that restoration of cytosolic purine nucleotide levels rescues the constitutive autophagy phenotype of rns2-2 seedlings, whereas inhibition of purine synthesis induces autophagy in wild-type seedlings. rns2-2 seedlings have reduced activity of the target of rapamycin (TOR) kinase complex, a negative regulator of autophagy, and this phenotype is rescued by addition of inosine to increase purine levels. Activation of TOR in rns2-2 by exogenous auxin blocks the enhanced autophagy, indicating a possible involvement of the TOR signaling pathway in the activation of autophagy in the rns2-2 mutant. Our data suggest a model in which loss of rRNA degradation in rns2-2 leads to a reduction in cytoplasmic nucleotide concentrations, which in turn inhibits TOR activity, leading to activation of autophagy to restore homeostasis.

Proteogenic Dipeptides Are Characterized by Diel Fluctuations and Target of Rapamycin Complex-Signaling Dependency in the Model Plant Arabidopsis thaliana

“…As in other organisms, plant TORC is an autophagy repressor by inhibiting AuTophaGy (ATG) genes ( Liu and Bassham, 2010 ) and possibly binding to the autophagy initiators ATG1-ATG13 ( Van Leene et al, 2019 ). When purine levels are reduced, TOR activity is decreased into Arabidopsis T2 family endoribonuclease RNS2 ( rns2-2 ) mutant, which in turn activates autophagy, suggesting that TOR mediates this catabolic process by responding to the levels of nucleotides to restore homeostasis ( Kazibwe et al, 2020 ). As a consequence, the autophagy-induced recycling of nutrients can reactivate the TOR pathway, while nucleotides promote TOR activity ( Busche et al, 2021 ).…”

Target of Rapamycin in Control of Autophagy: Puppet Master and Signal Integrator

“…Other TOR upstream signals such as sulfur [191], amino acids [209], and biotic and abiotic stress have all been implicated in regulation of autophagy in plants. Purine nucleotide depletion also activates autophagy in Arabidopsis via inhibition of TOR [224]. Overexpression of TOR and exogenous addition of the auxin NAA, an upstream activator of TOR, prevented autophagy activation in response to several abiotic stresses [19], confirming the importance of TOR in autophagy regulation in plants.…”

An active RNA transport mechanism into plant vacuoles

“…Vacuoles from ski2 mutant plants have compromised RNA transport and show the same constitutive autophagy phenotype as rns2 mutants that lack the main vacuolar RNase activity 6,8 . Moreover, altered nucleotide homeostasis in mutants with defective vacuolar RNA salvage can be compensated by feeding seedlings with purine nucleosides, and the rns2 constitutive autophagy phenotype is chemically complemented by inosine feeding in a dose-dependent manner 10 . Consistent with a role in the RNA salvage pathway, the ski2 autophagy phenotype is also complemented by inosine feeding, although the concentration of inosine needed for this complementation is higher than the concentration needed to eliminate the rns2 phenotype.…”

“…Autophagy was quantified in seedling roots by staining of acidic organelles with the lipophilic dye MDC and in leaf protoplasts by transient expression of the autophagosome marker GFP-ATG8e as previously described 56 . Chemical complementation of the ski2-5 constitutive autophagy phenotype was performed as in Kazibwe et al 10 , except that inosine was used at a final concentration of 50 µM. MDC-stained autophagosomes were counted per frame from at least 10 root images taken in the late elongation zone, while GFP-labeled structures were counted from at least 100 protoplasts with active autophagy.…”

“…Vacuolar RNA degradation is critical for the maintenance of nucleotide homeostasis 9,10 . Mutants with deficiencies in the nucleotide salvage pathway, including those with defects in vacuolar RNA degradation such as rns2 [6][7][8] , have constitutive activation of the autophagy pathway.…”