Regulation of autophagy through SnRK1 and TOR signaling pathways

ABSTRACT

Autophagy is important for degradation and recycling of cytoplasmic materials in all eukaryotes and is often triggered by environmental stress. How autophagy is activated in plants under different environmental conditions is still poorly understood. Our recent studies show that induction of autophagy by different abiotic stress conditions can occur via either a TOR-dependent or –independent pathway, depending on the stress. The SnRK1 protein kinase complex acts upstream of TOR in regulation of autophagy during nutrient deficiency, salt and osmotic stresses. In contrast, oxidative and ER stress regulate autophagy in a SnRK1-dependent but TOR-independent manner. Here we summarize and discuss these distinct pathways for activation of autophagy under different environmental stress conditions.

Autophagy functions in the degradation and recycling of cytoplasmic materials in the vacuole or lysosome in all eukaryotes.^1,2 In plants, autophagy is activated as a response to various abiotic and biotic stresses, during which it recycles nutrients and clears damaged cellular components.^3-8 A major regulator of autophagy is the target of rapamycin (TOR) protein kinase complex, which inhibits autophagy and activates growth.⁹ The TOR catalytic subunit functions as a complex with two binding partners, regulatory-associated protein of TOR (RAPTOR) and Lethal with Sec Thirteen 8 (LST8).^10,11

Since TOR has been shown in other organisms to regulate autophagy in response to nutrient conditions,¹¹ we hypothesized that nutrient deficiency-induced autophagy might be dependent on TOR, whereas activation of autophagy by other stresses might be TOR-independent. Our results showed that autophagy activation requires repression of TOR activity not only upon nutrient deficiency, but also during salt and osmotic stress.¹² Activation of autophagy by oxidative stress or endoplasmic reticulum (ER) stress is, by contrast, independent of TOR.¹²

We also identified a second protein kinase complex that positively regulates autophagy in response to stress, the Snf1-related protein kinase 1 (SnRK1) complex.^13,14 SnRK1 is a plant ortholog of AMP-activated protein kinase (AMPK) in animals, and sucrose nonfermenting 1 (Snf1) in yeast, and functions as an energy sensor in plant growth and development.^15,16 A knockout mutant in the SnRK1 catalytic subunit KIN10 was unable to properly activate autophagy under all abiotic stresses tested. However, the constitutive autophagy observed upon disruption of TOR signaling was not blocked in the kin10 mutant, suggesting that TOR acts downstream of KIN10, and that KIN10 may act as an upstream sensor of certain stress signals for TOR signaling¹³ (Fig. 1).

Figure 1.

A model of autophagy regulation upon stress exposure in plants. Autophagy is induced by different stress conditions through distinct pathways. Nutrient deficiency, salt and osmotic stress activate SnRK1, which negatively regulates the TOR complex. TOR inhibits autophagy through inhibition of ATG1 or other unknown downstream regulators. Osmotic stress can also induce autophagy through a SnRK1-independent pathway that inhibits the TOR complex. Oxidative stress induces autophagy through SnRK1 and ATG1, independently of TOR. ER stress induces autophagy through SnRK1 and IRE1b, and is TOR-independent. It is unclear whether SnRK1 and IRE1b regulate autophagy via two parallel pathways or within the same pathway, and their order of action is unknown. Arrows are color-coded according to the stress condition to which they apply. Solid arrows indicate pathways with experimental evidence, while dashed arrows indicate potential pathways that need future investigation.

Our data show that nutrient deficiency-, salt- and osmotic stress-induced autophagy is both KIN10 and TOR-dependent, suggesting that autophagy is regulated through a SnRK1-TOR signaling pathway. We therefore propose that the SnRK1 complex senses stress signals and represses TOR, which in turn allows activation of autophagy. AMPK, the KIN10 ortholog in mammals, inhibits TOR through phosphorylation of its binding partner RAPTOR to activate autophagy upon nutrient deficiency.¹⁷ In Arabidopsis, KIN10 has been shown to interact in vivo with RAPTOR and to phosphorylate RAPTOR in vitro,¹⁸ suggesting that SnRK1 phosphorylation of RAPTOR represses TOR complex activity to activate autophagy in plants (Fig. 1).

Osmotic stress-induced autophagy was repressed by overexpression of TOR, but was not fully blocked in the kin10 mutant, indicating an alternative pathway for sensing osmotic stress signals for activation of autophagy through TOR^12,13 (Fig. 1). In common with other environmental stresses, osmotic stress leads to increased reactive oxygen species (ROS) levels in plant cells, which also function as upstream signaling molecules for autophagy activation.¹⁹ However, we have previously shown that osmotic stress-induced autophagy is independent of NADPH oxidase, a major source of signaling ROS.⁵ Another candidate regulator is KIN11, which is an alternative isoform of KIN10, and has also been suggested to regulate stress responses with both overlapping and distinct functions compared with KIN10.^15,20 However, the addition of a SnRK1 inhibitor, which blocks both KIN10 and KIN11 activity, also failed to completely block autophagy under osmotic stress, suggesting that KIN11 is not responsible for the remaining autophagy observed in a kin10 mutant upon osmotic stress treatment. It appears likely that additional, yet unidentified stress signals and sensors that regulate autophagy exist upstream of TOR.

Both oxidative stress and ER stress induce autophagy through a TOR-independent but SnRK1-dependent pathway,^12,13 suggesting that SnRK1 can regulate autophagy through TOR and also through other parallel signaling pathways. In yeast and mammals, AMPK/Snf1 regulates autophagy through both inhibition of TOR and phosphorylation of the core autophagy component ATG1.^21-24 A recent study showed that overexpression of KIN10 can enhance phosphorylation of ATG1 and that KIN10 can interact with ATG1a and ATG13a in vitro,¹⁴ suggesting that SnRK1 can also regulate autophagy through the ATG1 complex (Fig. 1).

ER stress is triggered by the accumulation of unfolded and misfolded proteins caused by environmental stress conditions such as excessive heat,²⁵ and leads to activation of autophagy.^7,26 Our data suggested that activation of autophagy by ER stress is dependent on KIN10 but not on TOR signaling.^12,13 ER stress triggers the unfolded protein response (UPR) to aid proper folding of the misfolded proteins, and the splicing factor inositol-requiring enzyme-1 (IRE1) acts as an ER stress sensor for activation of UPR genes.²⁵ ER stress-induced autophagy requires one of the isoforms of IRE1, IRE1b.⁷ It is possible that SnRK1 and IRE1b act in two parallel pathways for autophagy induction upon ER stress, or that KIN10 and IRE1b are in the same pathway (Fig. 1); additional work is needed to distinguish between these possibilities.

In summary, our recent research has revealed that autophagy in plant cells is regulated through both SnRK1- and TOR-dependent, and SnRK1-dependent but TOR–independent pathways in response to different environmental stresses. Future investigations are required to identify unknown autophagy regulators and stress sensors of autophagy regulation in plants that are involved in the SnRK1 and TOR pathways.

Funding Statement

This work was supported by grants no. DE-SC0014038 from the United States Department of Energy and no. 1R01GM120316–01A1 from the United States National Institutes of Health to DCB.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.