Exploring the association between nucleotide homeostasis and TOR-mediated autophagy responses in rRNA turnover-defective Arabidopsis mutants

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Kazibwe, Zakayo
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Bassham, Diane C
Macintosh, Gustavo
Walley, Justin
Aung, Kyaw
Kelley, Dior
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Genetics, Development and Cell Biology
To maintain cell viability, organisms degrade aberrant, nonfunctional or old cell components and their constituents recycled, mostly through a non-selective autophagy pathway that occurs in the vacuole/lysosomes and activated during environmental stress. Ribosomes are complex macromolecules comprised of ribosomal protein and ribosomal RNA (rRNA), and their number and quality must be regulated to maintain gene expression and homeostasis. Significant progress has been made in deciphering, ribosome assembly defective rRNA decay, and quality control mechanisms in eukaryotes. However, the turnover of mature ribosomes at the end of their useful life is not well studied. The degradation of rRNA in plant vacuoles is mediated by T2 family endoribonuclease 2 (RNS2). Plants that are defective in RNS2 have constitutively high basal autophagy levels, implying that rRNA turnover is essential for maintaining homeostasis. The vacuolar degradation of rRNA results in nucleosides that are transported to the cytoplasm by equilibrative nucleoside transporter 1 (ENT1). Autophagy is controlled by several upstream regulators, one of which is Target of Rapamycin (TOR) that negatively regulates autophagy activity under optimal growth conditions in plants. TOR coordinates both environmental and intracellular signals to regulate plant metabolism. Although several signals such as nitrogen, amino acids, phosphorus, and stress have to some extent been evaluated to be upstream cues for TOR activation/repression, little is known about nucleotides related TOR signaling. TOR is known to synchronize nucleotide availability with processes such as ribogenesis, ensuring that such processes begin only when sufficient nucleotide and other resources are available. Vacuolar degradation of rRNA is a significant source of nucleotides that may contribute to cytosolic nucleotide pull and thus to the regulation of TOR-coordinated ribosome or nucleic acid biosynthesis and autophagy. In this dissertation, I focused on the regulation of autophagy in rns2 and ent1 and how this autophagy activation is related to nucleotide salvage and homeostasis. I discovered that autophagy is activated by depletion of nucleotides from WT seedlings, and it is blocked by resupplying nucleotides to these seedlings. The constitutive autophagy of both rns2 and ent1 is suppressed by supplementation of the mutant seedlings with nucleosides, particularly inosine. Further, these mutants exhibit a reduced TOR activity that is restored to WT levels by exogenous inosine supplementation. Taken together, the results reported here demonstrate that both ribosomal RNA degradation in the vacuole and recycling of the resulting nucleosides to the cytoplasm are each independently essential for maintaining nucleotide homeostasis. A defect in either RNS2 or ENT1 may cause low cytosolic nucleoside concentration and suppression of the activity of a master metabolic regulatory protein TOR, resulting in activation of autophagy, probably to recycle important nutrients for plant survival. My work revealed nucleotides as novel upstream signals in the TOR-mediated regulation of autophagy responses in Arabidopsis.