Nonuple atg8 mutant provides genetic evidence for functional specialization of ATG8 isoforms in Arabidopsis thaliana
Autophagy is a vital cellular process that maintains organismal health by recycling damaged or superfluous cellular components within autophagosomes. This process is mediated by conserved ATG proteins, which coordinate autophagosome biogenesis and selective cargo degradation. Among these, the ubiquitin-like ATG8 protein plays a central role by linking cargo to the growing autophagosomes through interactions with selective autophagy receptors. Unlike most ATG proteins, which are represented by single or few isoforms, the ATG8 gene family is expanded in vascular plants, yet the extent of functional specialization within ATG8 isoforms remains largely unknown. Using transcriptional and translational reporters in Arabidopsis thaliana, we revealed that ATG8 isoforms are differentially expressed across tissues and form distinct autophagosomes within the same cell. To explore ATG8 specialization, we generated the nonuple ?atg8mutant 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 the specialization of ATG8 isoforms in plants and lay a foundation for investigating their roles in diverse cell types and stress conditions. Overall design: For standard plant growth, Arabidopsis seeds were gas sterilized with sodium hypochlorite + HCl (10:1 v/v), sown on water-saturated soil and grown in 16h light/8h dark photoperiod with 165 µmol m^-2 s^-1 light intensity. For in vitrogrowth, Arabidopsis seeds were surface sterilized in 70% ethanol for 10 minutes twice, then rinsed in absolute ethanol and dried on sterile paper. Seeds were sown in ½ MS liquid medium (Murashige and Skoog salt + Gamborg B5 vitamin mixture [Duchefa] supplemented with 0.5 g/liter MES and 1% sucrose, pH 5.7), vernalized at 4 °C in the dark for 2 days, and then grown under LEDs with 85µM/m²/s with a 14 h light/10 h dark photoperiod.High-quality DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method and used to construct Illumina-compatible libraries with the Nextera XT DNA Library Preparation Kit, following the manufacturer's instructions. Sequencing was performed on an Illumina NextSeq instrument in paired-end 150 bp mode. Raw FASTQ files obtained from sequencing were quality-checked and adapter-trimmed using TrimGalore (https://github.com/FelixKrueger/TrimGalore) with default settings. The trimmed FASTQ files were aligned to the TAIR10 genome using Bowtie2 (Langmead & Salzberg, 2012) with the parameters -D 15 -R 2 -N 0 -L 22 -i S,1,1.15. The resulting aligned BAM files were sorted and indexed using SAMtools (Danecek et al., 2021) and manually inspected for insertions or deletions using the Integrative Genomics Viewer (IGV).