A tale of regulation: Analysis of the role of SnRK1 and glutathione in autophagy in Arabidopsis thaliana

Autophagy is a degradation process in which cells break down and recycle their cytoplasmic contents when subjected to environmental stress or during development. Upon activation of autophagy, a double membrane vesicle called the autophagosome forms around the cargo and delivers it to the vacuole/lysosome for degradation. In mammals, regulation of autophagy has been extensively studied. In plants, key regulators of autophagy have been identified, but their upstream components are poorly understood. This dissertation summarizes my efforts in studying the regulation of autophagy in Arabidopsis thaliana.\nAMPK in animals, and its yeast homolog Snf1, are positive regulators of autophagy. The SnRK1 complex is the plant ortholog of AMPK and is a protein kinase that senses changes in energy levels and triggers downstream responses to enable survival. Here I demonstrate that SnRK1 is a positive regulator of autophagy in plants. Overexpression of the SnRK1 catalytic subunit, KIN10, led to increased autophagy under nutrient rich conditions, indicating activation of autophagy by SnRK1. A kin10 mutant had a basal level of autophagy under control conditions similar to wild-type plants, but activation of autophagy by most abiotic stresses was blocked, indicating that SnRK1 is required for autophagy induction by a wide variety of stress conditions. In addition, epistasis analysis showed that SnRK1 is upstream of TOR, a negative regulator of autophagy.\nGlutathione is an antioxidant that serves as scavenger of reactive oxygen species to maintain cellular homeostasis. In mammals, glutathione has been linked to activation of autophagy, but in plants this has not been reported. I demonstrate that glutathione is\nrequired for the activation of autophagy during salt stress or nutrient starvation, but not during osmotic or oxidative stress. Furthermore, glutathione acts as a signal molecule to induce autophagy independent of reactive oxygen species. In addition, regulation of autophagy by glutathione acts upstream of TOR, most likely by regulating SnRK1 activity.\nThe RNS2 ribonuclease and autophagy participate in ribosomal turnover in Arabidopsis. Plants without RNS2 activity have constitutive autophagy. A chemical approach was used to test the SnRK1 complex and the TOR signaling pathway as possible regulators for the activation of the constitutive autophagy of rns2-2 mutant. Here I report that activation of the TOR signaling pathway represses the constitutive autophagy in rns2-2 mutant. Inhibition of the SnRK1 complex by trehalose-6-phosphate did not inhibit the constitutive autophagy in rns2-2 mutant, indicating that the activation of autophagy is independent of the SnRK1 complex. Activation of TOR kinase by auxin or brassinolide resulted in the inhibition of the autophagy activity in rns2-2 mutant, indicating that the TOR signaling pathway is involved in the activation of autophagy in rns2-2 mutant.\nIn summary, this dissertation demonstrates that upon salt stress and nutrient starvation, glutathione positively regulates autophagy, most likely through activation of SnRK1, a positive autophagy regulator upstream of TOR. The TOR signaling pathway is involved in the activation of autophagy in the rns2-2 mutant, possibly due to the inactivation of the TOR kinase.