Autophagy Deficiency Compromises Alternative Pathways of Respiration following Energy Deprivation in Arabidopsis thaliana

Abstract

Under heterotrophic conditions, carbohydrate oxidation inside the mitochondrion is the primary energy source for cellular metabolism. However, during energy-limited conditions, alternative substrates are required to support respiration. Amino acid oxidation in plant cells plays a key role in this by generating electrons that can be transferred to the mitochondrial electron transport chain via the electron transfer flavoprotein/ubiquinone oxidoreductase system. Autophagy, a catabolic mechanism for macromolecule and protein recycling, allows the maintenance of amino acid pools and nutrient remobilization. Although the association between autophagy and alternative respiratory substrates has been suggested, the extent to which autophagy and primary metabolism interact to support plant respiration remains unclear. To investigate the metabolic importance of autophagy during development and under extended darkness, Arabidopsis (Arabidopsis thaliana) mutants with disruption of autophagy (atg mutants) were used. Under normal growth conditions, atg mutants showed lower growth and seed production with no impact on photosynthesis. Following extended darkness, atg mutants were characterized by signatures of early senescence, including decreased chlorophyll content and maximum photochemical efficiency of photosystem II coupled with increases in dark respiration. Transcript levels of genes involved in alternative pathways of respiration and amino acid catabolism were up-regulated in atg mutants. The metabolite profiles of dark-treated leaves revealed an extensive metabolic reprogramming in which increases in amino acid levels were partially compromised in atg mutants. Although an enhanced respiration in atg mutants was observed during extended darkness, autophagy deficiency compromises protein degradation and the generation of amino acids used as alternative substrates to the respiration.

Figure 1.

Seed and silique phenotypes observed in Arabidopsis atg mutants. A, Number of seeds per silique. B, Seed weight. C, Silique length. D, Seed yield. E, Total siliques per plant. F, Branch number. Seed weight was obtained by measuring 500 seeds (n = 10). Silique length (C) was determined in images taken with a digital camera (Canon Powershot A650 IS) attached to a stereomicroscope (Zeiss Stemi 2000-C). The measurements were performed on the images using ImageJ software. Values presented are means ± se of at least 10 biological replicates per genotype. Asterisks designate values that were determined by Student’s t test to be significantly different (P < 0.05) from the wild type (WT).

Figure 2.

Phenotypes of Arabidopsis atg mutants under extended dark treatment. A, Images of 4-week-old, short-day-grown Arabidopsis plants immediately light was turned off (0 d) and after further treatment for 9 d in darkness conditions. B and C, Chlorophyll (Chl) content (B) and F_v/F_m (C) of leaves of 4-week-old, short-day-grown Arabidopsis plants after further treatment for 0, 3, 6, and 9 d in extended darkness. Values are means ± se of five independent samplings. Asterisks indicate values that were determined by Student’s t test to be significantly different (P < 0.05) from the wild type (WT) at each time point analyzed. FW, Fresh weight.

Figure 3.

Dark respiration during extended dark treatment. The CO₂ efflux rates of 4-week-old Arabidopsis plants immediately after light was turned off (0 d) and during further treatment for 9 d in darkness were analyzed. Gas-exchange measurements were performed with an open-flow infrared gas-exchange analyzer system with a portable photosynthesis system to fit a whole-plant cuvette. Values presented are means ± se of seven biological replicates per genotype. Asterisks designate values that were determined by Student’s t test to be significantly different (P < 0.05) from the wild type (WT).

Figure 4.

Metabolite levels in Arabidopsis atg mutants. Starch (A), protein (B), and amino acids (C) were measured using whole rosettes of 4-week-old short-day-grown Arabidopsis plants after further treatment for 0, 3, 6, and 9 d in extended darkness. Values presented are means ± se of five biological replicates per genotype. Asterisks designate values that were determined by Student’s t test to be significantly different (P < 0.05) from the wild type (WT) at each time point analyzed. FW, Fresh weight.

Figure 5.

Relative levels of sugars and organic acids in Arabidopsis atg mutants during extended dark conditions as measured by gas chromatography-mass spectrometry (GC-MS). The y axis values represent the metabolite level relative to the wild type (WT). Data were normalized relative to the mean content calculated for the 0-d dark-treated leaves of the WT (in case no response was detected at 0 d, normalization was performed against 3-d dark-treated leaves of the wild type). Values presented are means ± se of five biological replicates per genotype. Asterisks designate values that were determined by Student’s t test to be significantly different (P < 0.05) from the WT at each time point analyzed.

Figure 6.

Relative levels of amino acids in Arabidopsis atg mutants during extended dark conditions as measured by GC-MS. The y axis values represent the metabolite level relative to the wild type (WT). Data were normalized to the mean response calculated for the 0-d dark-treated leaves of the WT (in case no response was detected at 0 d, normalization was performed against 3-d dark-treated leaves of the wild type). Values presented are means ± se of five biological replicates per genotype. Asterisks designate values that were determined by Student’s t test to be significantly different (P < 0.05) from the WT at each time point analyzed.

Figure 7.

Changes in transcript levels in 4-week-old, short-day-grown Arabidopsis plants after further treatment for 0, 3, 6, and 9 d in extended darkness. Transcript abundance is shown for genes associated with the alternative pathways of respiration, including ETFβ (A), ETFQO (B), IVDH (C), D2HGDH (D), LKR/SDH (E), SAG12 (F), SAG13 (G), and CV (H). The y axis values represent the metabolite level relative to the wild type (WT). Data were normalized with respect to the mean response calculated for the 0-d dark-treated leaves of the wild type. Values presented are means ± se of three independent biological replicates. Asterisks indicate values that were determined by Student’s t test to be significantly different (P < 0.05) from the wild type at each time point analyzed.

Figure 8.

Schematic model showing the catabolic process involved in macromolecule degradation leading to electron donation to the ETF-ETFQO pathway during dark-induced senescence in wild-type plants (A) and atg mutants (B). Carbon starvation conditions promoted by extended darkness are associated with macromolecule degradation via several catabolic pathways (e.g. autophagy), releasing amino acids to be oxidized. The electrons generated are transferred, via the ETF/ETFQO system, to the respiratory chain through the ubiquinol pool, promoting plant survival. In atg mutants, there is a compromised amino acid (aa) supply, particularly BCAAs and Lys, recognized previously to be able to feed electrons to the ETF/ETFQO system. Simultaneously, there is a higher induction of genes associated with the ETF/ETFQO pathways and autophagy independent of chloroplast degradation, CV, which leads to a hypersensitivity response to energetic limitations in atg mutants.