An additional role for chloroplast proteins—an amino acid reservoir for energy production during sugar starvation

ABSTRACT

Autophagy is an evolutionarily conserved system that degrades intracellular components including proteins and organelles, and is important in the adaptive response to starvation in various eukaryotic organisms. Plant chloroplasts convert light energy into chemical energy and assimilate atmospheric carbon dioxide (CO₂) for carbohydrate production through photosynthesis reactions. We previously described an autophagy process for chloroplast degradation, during which a portion of chloroplasts are mobilized into the vacuole via autophagic vesicles termed Rubisco-containing bodies. Our recent study demonstrated that the activation of autophagy in photoassimilate-limited leaves is required for the production of free amino acids (AAs) as an alternative energy source. The catabolism of free branched-chain amino acids (BCAAs) is particularly important for survival under starvation conditions. These recent findings suggest an additional role for chloroplasts as a reservoir of AA when photosynthetic energy production is limited.

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Organisms require energy to survive and grow. Land plants primarily acquire energy via photosynthetic conversion of sunlight energy to chemical energy, which in turn fuels the assimilation of atmospheric CO₂ for carbohydrate production. Carbohydrates produced in chloroplasts become the major substrate for energy production in the cytoplasm and mitochondria through respiration. Arabidopsis (Arabidopsis thaliana) plants store these carbohydrates in the form of starch in chloroplasts during the day, and break these molecules down for respiratory energy production during the night.^1,2 This energy maintenance system based on starch production and breakdown can be perturbed by environmental stress.³ For instance, insufficient light due to suboptimal climate or shading by neighboring plants inhibits photosynthetic starch production, resulting in an energy shortage. Extreme temperature, drought and hypoxia also interfere with photosynthesis. During such energy-starved conditions, plants require alternative substrates for energy production.⁴

The majority of nutrients in photosynthetic tissues is present in chloroplast proteins, such that 75–80% of a C3 plant’s total nitrogen is found in the leaf chloroplasts.⁵ The CO₂-fixing enzyme Rubisco is especially prominent in this distribution, accounting for 10–30% of total leaf nitrogen.⁶ Therefore, when photosynthesis is inhibited, chloroplasts and photosynthetic proteins become attractive targets as alternative energy sources via catabolic reactions. Autophagy is the main intracellular process that degrades organelles and proteins to facilitate catabolic metabolism in eukaryotes. During autophagy, a portion of cytoplasm is sequestered by double-membrane bound vesicles termed autophagosomes for subsequent digestion in lytic organelles (i.e., vacuoles in plants or lysosomes in animals).⁷ Among the autophagy-related genes found in arabidopsis, AUTOPHAGY (ATG) 1–10, 12–14, 16, and 18 are required for autophagosome formation.⁸ We previously identified an autophagic process that degrades chloroplasts in rice and arabidopsis.^9,10 During this process, portions of the stroma and chloroplast envelope are transported into the vacuole in a type of autophagosome termed Rubisco-containing bodies (RCBs).¹¹ We further demonstrated the activation of RCB-mediated autophagy in energy-starved leaves,¹² and described its contribution to energy production in arabidopsis.¹³ Our recent study examines how autophagy supports energy production in mature leaves when starved.¹⁴

Using fluorescent protein markers that visualize chloroplast-targeted autophagy, we found that RCB production is activated in plants that are maintained in darkness for 2 days to induce sugar starvation.¹⁴ Consistent with this observation, wild-type plants showed a decrease in the amount of Rubisco and soluble proteins during treatment, while in autophagy-deficient mutants, such as atg5 and atg2, the decrease in protein content was suppressed. We also measured the level of free amino acids – products derived from protein degradation. Although the amount of free amino acids increased in leaves of sugar-starved plants overall, the increase in basic AAs (Lys, Arg and His), aromatic AAs (Phe, Tyr and Trp) and BCAAs (Val, Leu and Ile) was suppressed in the atg plants relative to wild type. Another study using a different set of autophagy-deficient mutants, atg5, atg7 and atg9, showed a lower increase in the levels of basic AAs, aromatic AAs and BCAAs in the atg plants than in wild-type plants during dark treatment.¹⁵ These findings suggest a vital role for autophagy in the production of free AAs in response to sugar shortage.

The amount of free basic AAs, BCAAs and aromatic AAs are extremely low in control plants grown without any stress treatment;¹⁶ however numerous metabolomic analyses report an increase in free basic AAs, BCAAs and aromatic AAs during energy-starved conditions.^17-20 During these conditions, free amino acids are degraded through catabolic enzyme cascades into simple carbon skeletons that can be integrated into the mitochondrial TCA cycle for respiratory energy production.¹⁶ Enzymes involved in BCAA catabolic reactions are essential for survival during extended darkness,^18-21 leading us to hypothesize that autophagy could be a major route to supply free BCAAs to this catabolic cascade. In accordance with this hypothesis, we measured the free amino acid levels in the double mutants of atg5 and BCAA catabolism-related genes ELECTRON TRANSFER FLAVOPROTEIN-UBIQUINONE OXIDOREDUCTASE (ETFQO) and ISOVALERYL-CoA DEHYDROGENASE (IVDH). The enhanced accumulation of free BCAAs in etfqo and ivdh single mutant lines was diminished by the addition of the atg5 mutation.⁷ These results suggest an amino acid recycling route during sugar shortage in which free BCAAs derived from active degradation of chloroplast proteins via autophagy are reused as an energy source through enzymatically catabolic cascades in the mitochondria (Figure 1).

Figure 1.

Schematic model of a route for energy production during an early stage of sugar starvation (around 2 d of dark treatment) in arabidopsis mesophyll cells. When photosynthetic sugar production is disrupted in wild-type plants, piecemeal degradation of chloroplast stroma via autophagy is activated to produce free amino acids. A portion of the released free amino acids, especially branched-chain amino acids (BCAAs), are reused for respiratory energy production through amino acid catabolism in the mitochondria. In autophagy-deficient mutants, this response is impaired, resulting in an uncontrolled chloroplast degradation such as the strong activation of chloroplast vesiculation-containing vesicles (CCVs)-mediated pathway,²² which is independent of autophagy, as previously suggested.¹⁵

In the double mutants of etfqo atg5 and ivdh atg5 plants, the accumulation of free BCAAs was not completely suppressed during dark treatment, indicating that autophagy-independent proteolysis contributes to the production of free BCAAs. Degradation of ubiquitinated proteins via cytoplasmic 26S proteasome is a ubiquitous protein turnover system in eukaryotes. 26S proteasomes are specifically protected from autophagic turnover during sugar starvation;^23,24 therefore, the ubiquitin-proteasome system may be another major route to produce free AAs in response to sugar shortage.

Unlike the chloroplast stroma that was actively transported into the vacuolar lumen via autophagy during dark treatment, fluorescent protein-labeled mitochondria from wild-type and atg5 plants showed no clear difference after 2 d of dark treatment.¹⁴ We previously described another type of chloroplast-targeted autophagy termed chlorophagy, during which unnecessary chloroplasts are selectively eliminated in their entirety.^25-27 Notably, chlorophagy did not occur in plants that were dark treated for only 2 d.¹⁴ Overall, our results lead us to conclude that the chloroplast stroma might be a preferential target (compared with mitochondria and whole chloroplasts) to produce free AAs as an alternative to sugars.

The current study focuses on plant responses to dark treatment, which is a simple experimental system with which to induce sugar starvation. In nature, sugar starvation is likely caused by weak light or abiotic stresses that interfere with photosynthesis; therefore, future studies should address the roles of chloroplast stroma-targeted autophagy during such stress conditions to better understand the survival strategies of plants. Numerous studies suggested the importance of autophagy in plant responses to various abiotic stresses, as well overviewed in a recent review.²⁸ For instance, autophagy supports plant survival during drought or submergence stress that limits photosynthetic and respiratory energy production.^29,30 Thus, the enhancement of autophagy activity may be an avenue to improve the plant tolerance to such stresses.

Funding Statement

This work was supported, in part, by Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Numbers 17H05050 and 18H04852 to M.I. and 15H04626 to H.I.), Japan Science and Technology Agency (JST) PRESTO (Grant Number JPMJPR16Q1 to M.I.), and the Program for Creation of Interdisciplinary Research at Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Japan (to M.I.).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.