Autophagy is required for lipid homeostasis during dark-induced senescence

Abstract

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon-starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) subjected to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming toward lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants.

Figure 1

Differential lipid response of atg mutants under dark-induced senescence. PCA was performed using the lipid data obtained in samples from 4-week-old Arabidopsis plants immediately before lights were turned off (0 day) and after further treatment for 6 d in darkness. Three loss-of-function mutants of the autophagy pathway, namely atg5-1, atg7-2, and atg9-4, and their WT were analyzed.

Figure 2

atg mutants present altered phospholipid levels under control and extended darkness conditions. Hierarchical clustering of (A) PC and (B) PE. Values plotted are log₂ fold change. Distances were measured by Pearson correlation using MeV 4.9.0 software. Data were normalized to the mean response calculated for the 0-day dark-treated leaves of the WT. Values presented are means ± SE of five to six biological replicates per genotype; an asterisk (*) designates values that were determined by Student’s t test to be significantly different (P < 0.05) from WT at each time point analyzed.

Figure 3

Chloroplast lipids are substantially reduced in atg mutants during dark-induced senescence. MGDG and DGDG values plotted are log₂ fold change. Data were normalized to the mean response calculated for the 0-day dark-treated leaves of the WT. Values presented are means ± SE of five to six biological replicates per genotype; an asterisk (*) designate values that were determined by the Student’s t test to be significantly different (P < 0.05) from WT at each time point analyzed.

Figure 4

Distinct accumulation of neutral lipids in atg mutants during dark-induced senescence. A, TAG and B, DAG. Values plotted are log₂ fold change. Data were normalized to the mean response calculated for the 0-day dark-treated leaves of the WT. Values presented are means ± SE of five to six biological replicates per genotype; an asterisk (*) designates values that were determined by Student’s t test to be significantly different (P < 0.05) from WT each time point analyzed.

Figure 5

Autophagy deficiency affects LD dynamics during dark-induced senescence. A, Representative confocal images of LDs (BODIPY 493/503 fluorescence, false color [green] in WT, atg5-1, and atg7-2 leaves). B, Merged image of BODIPY and chloroplast autofluorescence (false color red). Images were collected at the same magnification and are projections of Z-stacks of 20 optical sections. Bar = 10 μm. C, Number of total LDs per image area. The number of LDs was quantified by ImageJ as BODIPY‐stained lipid area in 2-D projections of Z‐stacks of three images of four biological replicates per genotype and time point.

Figure 6

PG-related genes are up-regulated in atg mutants during dark-induced senescence. Transcript abundance is shown for genes associated with PG, including PES-1 (A), PES-2 (B), ABC1K7 (C), and PPH (D). The y-axis values represent the gene expression level relative to the WT. Data were normalized with respect to the mean response calculated for the 0-day dark-treated leaves of the WT. Values presented are means ± SE of three independent biological replicates. An asterisk (*) indicates values that were determined by Student’s t test to be significantly different (P < 0.05) from the WT at each time point analyzed. A hash (#) indicates values determined by Student’s t test to be significantly different (P < 0.05) from WT at 0 day.

Figure 7

Autophagy disruption triggers altered PG response. A, Transmission electron micrographs of leaves from 4-week-old Arabidopsis plants immediately before lights were turned off (0 day) and after further treatment for 7 d of darkness. Scale bar = 1 μm. Quantification of the number (B) and size (C) of PG per chloroplasts (±SE, n = 6–7 chloroplasts of three independent cells); Analysis shown in A–C was performed in atg5-1 and its respective WT. D, Immunoblot analysis of the PG protein, FBN1a, was performed in leaves from 4-week-old Arabidopsis atg5-1, atg7-2, and WT plants immediately before (0 day) and after further treatment for 3 and 6 d in darkness. FBN1a protein displayed a predicted molecular mass of ∼30–35 kD. The detection of a second lower band in the region of 30 kD possibly corresponds to FBN1b protein (with high sequence similarity with FBN1a), as previously observed (GiacomelLi et al., 2006; Martinis et al., 2013, 2014).

Figure 8

Changes in transcript levels in WT and atg mutants during dark-induced senescence. Transcript abundance is shown for genes associated with lipid catabolic pathways, including ACX-4 (A), CSY-1(B) KAT-2 (C), MFP-2 (D), pMDH-1 (E), SDP-1 (F). The y-axis values represent the expression 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 WT. Values presented are means ± SE of three independent biological replicates. An asterisk (*) indicates values that were determined by Student’s t test to be significantly different (P < 0.05) from the WT at each time point analyzed. A number sign (#) indicates values determined by Student’s t test to be significantly different (P < 0.05) from WT at 0d.

Figure 9

Changes in FFA levels in WT and atg mutant plants under dark-induced senescence. Values plotted are log₂ fold change. Data were normalized to the mean response calculated for the 0-day dark-treated leaves of the WT. Values presented are mean ± SE of five to six biological replicates per genotype; an asterisk (*) designates values that were determined by the Student’s t test to be significantly different (P < 0.05) from WT each time point analyzed.

Figure 10

Schematic model of lipid remodeling response of atg mutants during extended darkness. Under extended darkness, the plastidial and extraplastidial membranes, composed by MGDG/DGDG and phospholipids, respectively, are degraded, releasing FAs to be stored as TAGs in LD in the cytosol. LDs and transient FFA donate substrates for β-oxidation in peroxisomes. Meanwhile, in atg mutants, there is a substantial degradation of chloroplast lipids, whereas the production of cytosolic LDs is impaired. Part of the released plastidial FAs, together with phytol derived from chlorophyll degradation, are channeled to the production of PG in the chloroplast. β-Oxidation pathways are highly induced to consume overloaded plastidial FFA and, possibly, PG lipids (question mark). The figure was created using BioRender (https://biorender.com).