Autophagy-related (ATG) 11, ATG9 and the phosphatidylinositol 3-kinase control ATG2-mediated formation of autophagosomes in Arabidopsis.

KEY MESSAGE: Using quantitative assays for autophagy, we analyzed 4 classes of atg mutants, discovered new atg2 phenotypes and ATG gene interactions, and proposed a model of autophagosome formation in plants. Plant and other eukaryotic cells use autophagy to target cytoplasmic constituents for degradation in the vacuole. Autophagy is regulated and executed by a conserved set of proteins called autophagy-related (ATG). In Arabidopsis, several groups of ATG proteins have been characterized using genetic approaches. However, the genetic interactions between ATG genes have not been established and the relationship between different ATG groups in plants remains unclear. Here we analyzed atg2, atg7, atg9, and atg11 mutants and their double mutants at the physiological, biochemical, and subcellular levels. Involvement of phosphatidylinositol 3-kinase (PI3K) in autophagy was also tested using wortmannin, a PI3K inhibitor. Our mutant analysis using autophagy markers showed that atg7 and atg2 phenotypes are more severe than those of atg11 and atg9. Unlike other mutants, atg2 cells accumulated several autophagic vesicles that could not be delivered to the vacuole. Analysis of atg double mutants, combined with wortmannin treatment, indicated that ATG11, PI3K, and ATG9 act upstream of ATG2. Our data support a model in which plant ATG1 and PI3K complexes play a role in the initiation of autophagy, whereas ATG2 is involved in a later step during the biogenesis of autophagic vesicles.

Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth.

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons for seedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of the seedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomal proteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents to the lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedling growth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in the vacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-type vacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, we propose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.

A revised assay for monitoring autophagic flux in Arabidopsis thaliana reveals involvement of AUTOPHAGY-RELATED9 in autophagy.

Autophagy targets cytoplasmic cargo to a lytic compartment for degradation. Autophagy-related (Atg) proteins, including the transmembrane protein Atg9, are involved in different steps of autophagy in yeast and mammalian cells. Functional classification of core Atg proteins in plants has not been clearly confirmed, partly because of the limited availability of reliable assays for monitoring autophagic flux. By using proUBQ10-GFP-ATG8a as an autophagic marker, we showed that autophagic flux is reduced but not completely compromised in Arabidopsis thaliana atg9 mutants. In contrast, we confirmed full inhibition of autophagic flux in atg7 and that the difference in autophagy was consistent with the differences in mutant phenotypes such as hypersensitivity to nutrient stress and selective autophagy. Autophagic flux is also reduced by an inhibitor of phosphatidylinositol kinase. Our data indicated that atg9 is phenotypically distinct from atg7 and atg2 in Arabidopsis, and we proposed that ATG9 and phosphatidylinositol kinase activity contribute to efficient autophagy in Arabidopsis.