Arabidopsis Vacuolar protein sorting 9a (<scp>VPS9a</scp>) is required for glutamine synthetase/glutamate synthase (<scp>GS</scp>/<scp>GOGAT</scp>) cycle and autophagy under nutrient starvation

Plants have developed complex endomembrane systems in response to environmental challenges such as nutrient deficiency. This study focused on the role of Vacuolar protein sorting 9 (VPS9a), a key regulatory gene involved in the endosomal sorting process in Arabidopsis thaliana. Loss of VPS9a function results in stress-sensitive phenotypes under carbon and nitrogen starvation. First, we investigated the changes in the Glutamine Synthetase/Glutamate Synthase (GS/GOGAT) cycle under nitrogen starvation and conducted a co-expression network analysis based on transcriptomic profiles. These results indicate that the endocytic pathway and the majority of the degradation processes are related to GS and NADH-GOGAT activity. Genes related to autophagy and endocytic pathways showed diverse response trends in Col-0, vps9a-2, and 35S:VPS9a-GFP/vps9a-2. Several autophagy- and endocytosis-related genes, including Autophagy-related protein 1 (ATG1), Autophagy-related protein 8 (ATG8), Thylakoid lumen protein (TLP18.3), Autoinhibited Ca(2+)-ATPase, Isoform 4 (ACA4), MAP kinase 2 (AtMKK2), and Extensin 21 (EXT21), were identified as hub genes. Further, we found that the loss of VPS9a function leads to reduced accumulation of autophagic bodies and a marked decrease in ATG8a protein levels but does not affect autophagic flux or the accumulation of ATG8 with phosphatidylethanolamine (PE). Interestingly, VPS9a appears to exert differential effects on various ATG8 Homologs. In summary, our results established a connection between autophagy, endocytic pathways, and nitrogen metabolism processes, identifying key hub genes involved in these processes. Among these hub genes, VPS9a was found to affect ATG8a levels, suggesting that VPS9a selectively regulates specific ATG8 proteins involved in autophagic processes.