Study of autophagy in higher plant - through the analysis of AtAPG genes -

Autophagy is an intracellular process for vacuolar bulk degradation of cytoplasmic components．Autophagy is a ubiquitous eukaryotic process and recently, the molecular machinery responsible for yeast and mammalian autophagy has begun to be elucidated at the cellular level．However, the role that autophagy plays at the organismal level are yet to be determined．Therefore, I aimed to study the significance of autophagy in plant for the plant life cycle．A genome wide homology search revealed significant conservation between yeast and plant autophagy genes．Twenty-five novel plant genes (AtAPG genes) were discovered, which are homologous to 12 yeast APG genes that are essential for autophagy．Both the functional domains and the amino acid residues essential for yeast autophagy are well conserved in the corresponding AtAPG proteins, suggesting that the Apg system functions in a similar manner in plants as it does in yeast and mammalian cells．Next, I searched for AtAPG knockout plants in T-DNA insertion lines and identified an Arabidopsis thaliana mutant carrying a T-DNA insertion within AtAPG9, which is the only homologue of yeast Apg9 in Arabidopsis (atapg9-1)．AtAPG9 was predicted to be a transmembrane protein, and reasonably AtAPG9-GFP showed the fluorescence of dots dispersed throughout the cytoplasm．AtAPG9 was expressed in every wild-type organ tested, and not in the atapg9-1 mutant plants．In the Arabidopsis suspension-cultured cells and root cells that during sucrose starvation, addition of protease inhibitor induced the accumulation of spherical bodies．These spherical bodies were stained with acidtropic dyes quinacrine or neutral red, this phenomena is quite similar to autophagy reported in tobacco suspension cells．This accumulation of spherical bodies was reduced in atapg9-1, suggesting that autophagy was defected in atapg9-1．Next, I observed the phenotypes brought about by the null mutation of AtAPG9 gene．The following phenotypes were complemented by expression of wild-type AtAPG9 gene, confirming that AtAPG9 is responsible for them．Under nitrogen starvation conditions, rapid chlorosis was observed in atapg9-1 cotyledons and rosette leaves． Under carbon starvation conditions, atapg9-1 also exhibited rapid chlorosis of cotyledons．Hence, in the nutrient limited condition, autophagy is crucial for retarding chlorosis．Furthermore, atapg9-1 exhibited a reduction in seed sets when nitrogen supply was limited, suggesting that AtAPG9 is required for efficient nutrient relocalization in as a whole plant．In detached leaves, nutrient supply to leaves was cut off and senescence was induced artificially．Detached leaves of atapg9-1 showed accelerated senescence．Microscopic analysis indicated that there was a reduction in the number of chloroplasts in the epidermal side of wild-type leaf mesophyll cells during senescence．Though, this reduction in the number of chloroplasts was also observed in atapg9-1, the loss of chlorophyll from each chloroplast was accelerated．In addition to that, morphology of the mesophyll cells was apparently different between wild-type and atapg9-1 at 6 days after detachment．This result suggests that AtAPG9 is required for proceeding the leaf senescence process properly．Even under nutrient growth conditions, the timing of bolting was accelerated in atapg9-1 plants．I confirmed that the slight nitrogen starvation accelerated the timing of bolting in Arabidopsis．Considering together, even though enough nutrient is supplied, loss of AtAPG9 will cause the slight nitrogen starvation in plant．These results indicate that autophagy is required for maintenance of the cellular viability during nutrient-limited conditions and leaf senescence．Efficient nutrient utilization within a whole plant can be achieved through such a vacuolar degradation process within cells