An A. thaliana mutant lacking all nine ATG8 isoforms provides genetic evidence for functional specialization of ATG8 in plants

Abstract

Autophagy sustains cellular health by recycling damaged or excess components through autophagosomes. Autophagy is mediated by conserved ATG proteins, among which the ubiquitin-like ATG8 proteins play a central role by linking cargo to the growing autophagosomes. Unlike most ATG proteins, the ATG8 gene family is significantly expanded in vascular plants, but its functional specialization remains poorly understood. Using transcriptional and translational reporters in Arabidopsis thaliana, we revealed that ATG8 isoforms are differentially expressed across tissues and form distinct autophagosomes. To explore ATG8 specialization, we generated the nonuple Δatg8 mutant, lacking all nine ATG8 isoforms. The mutant displayed hypersensitivity to carbon and nitrogen starvation, coupled with defects in bulk and selective autophagy, as shown by biochemical and ultrastructural analyses. Complementation experiments demonstrated that ATG8A could rescue both carbon and nitrogen starvation phenotypes, whereas ATG8H could only complement carbon starvation. Proximity labeling proteomics further identified isoform-specific interactors under nitrogen starvation, underscoring their functional divergence. These findings provide genetic evidence for functional specialization of ATG8 isoforms in plants and lay the foundation for investigating their roles in diverse cell types and stress conditions.

Keywords: Arabidopsis; Atg8; Autophagy; Selective autophagy.

Fig. 1.

Arabidopsis thaliana ATG8 isoforms exhibit tissue-specific expression patterns and form distinct autophagosomes within root cells. (A) Representative GUS staining images showing the spatial-temporal expression patterns of nine Arabidopsis ATG8 isoforms. 10-day-old Arabidopsis seedlings expressing pATG8X::GFP-GUS (X represents the nine ATG8 isoforms, from A to I) were stained with GUS staining buffer. Scale bar: 100 μm. (B) Representative confocal microscopic images showing the colocalization of mCherry–ATG8E with different GFP–ATG8 isoforms in Arabidopsis root epidermal cells. 5-day-old Arabidopsis seedlings co-expressing mCherry–ATG8E with GFP–ATG8E, GFP–ATG8A, GFP–ATG8D or GFP–ATG8I were incubated in ½ MS liquid medium containing either DMSO (as mock condition) for 2 h, 5 μM Torin 1 for 2 h, or 10 μg/ml tunicamycin for 4 h. Representative images of 10 replicates are shown here for A and B. Scale bars: 5 μm (main images); 3 μm (magnifications). (C) Quantification of mCherry–ATG8E colocalization ratio for the Arabidopsis root epidermal cells imaged in B. The mCherry–ATG8E colocalization ratio was calculated as the ratio of the number of mCherry–ATG8E puncta that colocalized with GFP–ATG8 isoforms to the total number of mCherry–ATG8E puncta. Bars indicate the mean±s.d. of 10 replicates. Brown–Forsy and Welch ANOVA tests with Dunnett's T3 multiple comparisons tests were used for statistically comparing the colocalization difference between each treatment group, with groups different from other groups at P<0.05 indicated by letters.

Fig. 2.

The atg8 nonuple mutant Δatg8 is deficient in autophagy. (A) Schematic representation of gene models illustrating CRISPR-induced mutations in the ATG8 gene. Blue arrows denote insertion sites; red arrows indicate deletion sites. The forward and reverse arrows within the gene model indicate the directionality of gene transcription, reflecting the strandedness of the gene. (B,C) Carbon (B) and nitrogen (C) starvation phenotypic assays comparing Col-0, atg5 and Δatg8 (n=3) in carbon-rich (+C) or carbon-deficient (−C) ½ MS liquid medium and nitrogen-rich (+N) or nitrogen-deficient (−N) ½ MS liquid medium. (D–F) Western blots comparing endogenous NBR1 levels in Col-0, atg5 and Δatg8 upon carbon (D) and nitrogen (F) starvation, in combination with concanamycin A (1 μM). Relative quantification of protein bands is reported in boxplots (E,G), representing the calculated values for three biological replicates. Boxes range from the 25% to the 75% percentiles, the horizontal lines correspond to the median and whiskers indicate the 5% and 95% percentiles.

Fig. 3.

The Δatg8 mutant is not able to perform mitophagy and pexophagy. (A) Western blots comparing endogenous IDH and VDAC levels in Col-0, atg5 and Δatg8 upon DNP treatment. (B,C) Relative protein levels of IDH (B) and VDAC (C) are represented in boxplots reporting the calculated values of three biological replicates. (D) Western blots comparing endogenous catalase levels in Col-0, atg5 and Δatg8 upon nitrogen starvation treatment (−N). Nitrogen-rich medium, +N. (E) Relative catalase protein levels, calculated from three biological replicates. (F) Electron micrographs of Col-0 and Δatg8 root cells treated with DNP or DMSO. Images representative of more than 30 sections from three individual roots. Scale bars: 500 nm. For all boxplots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 5th–95th percentiles.

Fig. 4.

Complementation of Δatg8 with ATG8A or ATG8H reveals functional specialization of ATG8 isoforms. (A,B) Carbon (A) and nitrogen (B) starvation phenotypic assays comparing Col-0, Δatg8 and complementation lines Δatg8 /+ATG8A, Δatg8/+ATG8H (n=3) in carbon-deficient (−C) ½ MS liquid medium and nitrogen-deficient (−N) ½ MS liquid medium. Images representative of two repeats. (C,E,G,I) Western blots comparing endogenous NBR1 levels in Col-0, Δatg8, Δatg8/+ATG8A (C,G) and Δatg8 /+ATG8H (E,I) upon carbon (C,E) and nitrogen (G,I) starvation, in combination with concanamycin A (1 μM). (D,F,H,L) Relative quantification of protein bands is reported in boxplots next to the corresponding western blot, representing the calculated values for three biological replicates. (M) Schematic representation of TurboID proximity labeling analysis. (M) Venn diagram reporting common and unique interactors of ATG8A and ATG8H under nitrogen starvation. For all boxplots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 5th–95th percentiles.