Chlorophagy does not require PLANT U-BOX4-mediated ubiquitination

ABSTRACT

Chloroplasts and mitochondria serve as intracellular energy production sites that are powered by the electron transport chain in their membranes. These organelles constantly accumulate damage, as their energetic reactions generate reactive oxygen species. To prevent the accumulation of damaged organelles and perturbation of cellular homeostasis, eukaryotic cells must remove damaged mitochondria and chloroplasts. Autophagy is the main route by which organelles are degraded. A type of mitochondrion-targeted autophagy known as mitophagy removes damaged mitochondria in mammalian cells; dysfunctional mitochondria that lose their membrane potential are marked by protein ubiquitination, becoming targets of selective mitophagy. Studies of the quality control system for chloroplasts in plants revealed the involvement of both autophagy and ubiquitination in the degradation of damaged chloroplasts. We recently assessed the relationship between chloroplast-associated ubiquitination mediated by PLANT U-BOX4 (PUB4) and chloroplast-targeted autophagy (chlorophagy) in the turnover of oxidatively damaged chloroplasts. Multiple assays using an Arabidopsis thaliana mutant revealed that PUB4-associated ubiquitination is dispensable for the induction of chlorophagy. Here, we describe the parallel functions of PUB4 and chlorophagy in chloroplast turnover and plant growth.

Mitochondria produce most of the energy for the growth of heterotrophic organisms through respiration. To produce ATP, mitochondria form a transmembrane potential across electron transport chains in their inner membranes. Leakage of electrons results in the accumulation of reactive oxygen species (ROS), leading to mitochondrial dysfunction. Since the accumulation of dysfunctional mitochondria impairs cell functions, eukaryotic cells must remove the damaged mitochondria.¹

Autophagy is an evolutionarily conserved process that mediates the transport of cytoplasmic materials into the lytic vacuole in yeast and plants and the lysosomes in animals.^2,3 The lytic organelles digest these cytoplasmic materials, allowing the reuse of their constituents such as amino acids and lipids. Macroautophagy is the major autophagy process, in which a double-membrane-bound vesicle termed the autophagosome sequesters a portion of the cytoplasm and delivers it into the vacuole/lysosomes.³ An autophagosome can engulf specific organelles; for example, autophagosomes can selectively eliminate damaged mitochondria in a process termed mitophagy.⁴ Autophagy is the main mechanism by which damaged mitochondria are removed to maintain cellular homeostasis in yeast and mammals.^1,5

Mitophagy is a well-studied process controlled by a post-transcriptional protein modification termed ubiquitination, in which small polypeptide ubiquitins are activated by E1 ubiquitin-activating enzymes, conjugated to E2 ubiquitin-conjugation enzymes, and then transferred to target proteins via E3 ubiquitin ligases.⁶ The 26S proteasome complex normally shreds ubiquitinated proteins in the cytoplasm. E3 ubiquitin ligases have specific targets; thus, the ubiquitin proteasome system enables highly controlled, selective protein degradation. In mammals, ubiquitination of mitochondrial outer-membrane proteins by cytoplasmic E3 Parkin induces their sequestration by autophagosomes.^1,7,8 Accumulation of the mitochondrion-targeted kinase PTEN-induced kinase 1 (PINK1) on the surface of depolarized mitochondria and PINK1-mediated phosphorylation of Parkin and ubiquitins acts as the trigger for ubiquitination of mitochondria.^9,10 When mitochondria are healthy and maintain transmembrane potential, PINK1 is imported into the mitochondria and degraded; therefore, mitophagy only causes the degradation of depolarized mitochondria. Ubiquitination of mitochondria is a key signal for inducing mitophagic removal of the depolarized mitochondrial fraction in mammalian cells.

Plants acquire energy through photosynthesis within chloroplasts. Electron transport chains in chloroplast thylakoid membranes produce a proton motive force using light energy; however, similar to mitochondrial inner membranes, electron leakage also occurs, generating ROS. Chloroplasts are therefore the major site of oxidative damage in plant leaves, and this damage, termed photoinhibition, is further aggravated when leaves are exposed to intense light. We previously identified an autophagy pathway termed chlorophagy, which transports photodamaged chloroplasts (Figure 1a).¹¹ This pathway selectively removes swollen chloroplasts associated with membrane damage from intense visible light via another type of autophagy-related membrane dynamic, termed microautophagy (Figure 1a),¹² during which the vacuolar membrane directly mediates the sequestration of degradation substrates.¹³ Another study found that proteins in chloroplasts hyperaccumulating ROS are ubiquitinated by a cytoplasmic E3 PLANT U-BOX4 (PUB4) for digestion (Figure 1b).¹⁴ Therefore, we postulated that chloroplasts with proteins ubiquitinated by PUB4 are selectively degraded by chlorophagy. We recently investigated this possibility by preparing mutant lines of PUB4 expressing fluorescent marker proteins to visualize the progression of chlorophagy.¹⁵ Evaluation of chlorophagy activity using biochemical assays and microscopy observations consistently showed similar chlorophagy activity in wild-type plants and the pub4 mutant line, pub4-6, when exposed to photoinhibition treatment. Therefore, we concluded that the induction of chlorophagy does not require the ubiquitination of chloroplast proteins mediated by PUB4 (Figure 1).¹⁵ We also observed no effect of pub4-6 mutation to the induction of another type of chloroplast autophagy that degrades the portion of chloroplast stroma as a specific cargo of autophagosomes termed Rubisco-containing body during carbon starvation.¹⁵ Although previous studies reported that autophagosomes deliver thylakoidal components and starch granules for degradation,^16,17 the occurrence of these pathways in pub4-6 plants has not been addressed.

Figure 1.

Schematic model for the parallel functions of chlorophagy and PLANT U-BOX4-mediated chloroplast degradation. (a) When Arabidopsis plants are subjected to photoinhibition resulting from exposure to intense light, swollen chloroplasts associated with membrane damage appear. An autophagy process termed chlorophagy transports such chloroplasts into the vacuole via a type of microautophagic membrane dynamic, which requires ATG5 and ATG7 functions. Core autophagy protein-related membrane and the vacuolar membrane mediate the incorporation of chloroplasts into the vacuole, although how these membranes cooperate remains uncertain. Unknown proteins or E3 ligase might act as a bridge between swollen chloroplasts and autophagy-related membranes during chlorophagy. (b) When chloroplasts overaccumulate a type of ROS, singlet oxygen (¹O₂), the cytoplasmic E3 PLANT U-BOX 4 (PUB4) ubiquitinates unknown chloroplast proteins and induces the lytic degradation of chloroplasts. This ubiquitination system is independent of the chlorophagic pathway described in (a). The PUB4-mediated system is especially induced during conditional ¹O₂ accumulation in plastid ferrochelatase2 (fc2) mutant plants. How ubiquitinated chloroplasts are mobilized to the vacuole has not been clearly characterized. Ub, ubiquitin

We further generated double mutant lines of PUB4 and genes essential for the production of the autophagosomal membrane, AUTOPHAGY5 (ATG5) or ATG7. The double mutants (pub4-6 atg5 or pub4-6 atg7) showed phenotypes synergistic with those of the respective single mutants under some conditions.¹⁵ During developmental growth, leaves of double mutants accumulated more ROS than wild-type or single-mutant plants, resulting in early cell death. The double mutation led to impaired seed production. Additionally, the double mutants were more susceptible to nitrogen or carbon starvation conditions, during which protein degradation and the subsequent recycling of small molecules are activated.^18,19 This genetic evidence also indicates that PUB4 and autophagy work independently under several conditions, and thus the loss of both systems in the double mutants leads to more severe phenotypes compared with the respective single mutants.

Our recent study clearly indicates that chlorophagy for photodamaged chloroplasts does not require PUB4 function.¹⁵ Identifying the molecules that trigger chlorophagy is an important goal in plant autophagy research. How plants orchestrate multiple protein degradation systems that function in parallel is a key question for understanding the survival strategy of land plants.

Funding Statement

This work was supported, in part, by Japan Society for the Promotion of Science (JSPS) KAKENHI [grant numbers, JP17H05050, JP18H04852, JP19H04712, JP20H04916, and JP20K21322 to M.I.; JP19J01681, JP20K15501, and JP20H05352 to S.N.], the JSPS Research Fellowship for Young Scientists [to S.N.], and Japan Science and Technology Agency (JST) PRESTO [grant number JPMJPR16Q1 to M.I.].

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.