Mechanism Exploration Of V-ATPase Subunit C Involved In Autophagy In Arabidopsis Thaliana

Autophagy is a lysosomal and vacuole-dependent degradation pathway,which is highly conserved in eukaryites.Once autophagy occurs in plant cells,the targets were encapsulated in the autophagosomes with double membrane,under the catalysis of the autophagy related proteins.And then autophagosomes were transported to vacuole for degradation.The degradation products were recycled.Autophagy is a intracellular degradation pathway highly complex and precisely.Under abiotic stress such as nutrient deficiency,drought and high salt,or biotic stress including pathogen invasion,cell recyling the intracellular macromolecules and damaged organelles to maintain the growth,development and survival of plant by autophagy.Autophagy can induce vacuolar acidification to ensure the activity of hydrolytic enzymes required for autophagosome degradation in yeast.Similar to yeast,V-ATPase is a type of proton pump located in the secretory membrane system in plant.The main function of the V-ATPase is catalyze ATP hydrolysis generating energy and transport hydrogen ion,at the same time acidify the vacuole.ConcanamycinA(ConcA)is a special inhabitor of V-ATPase.Previous study had show that ConcA can suppress processes of endocytosis,secretion and autophagosome degredation.It is suggest that V-ATPase plays an important role in autophagy pathway.V-ATPase is a complex including 12 subunits in Arabidopsis thaliana.At present,the physiological and biochemical functions of individual subunits are still unclear in autophagy pathway.The subunit C of V-ATPase(AtVHA-C)is encoded by a single gene in Arabidopsis thaliana.det3-1 is a mutant of AtVHA-C.Acidification ablity of TGN/EE is decreased,and transport process is delayed from TGN to vacuole,but acidification ablity of Golgi apparatus and vacuole are unaffected in det3-1.Previous study have found that det3-1 maybe an autophagy defective mutant.In this paper,we studied the tissue localization and subcellular localization of AtVHA-C systematacially.And explored the physiological function and mechanism of det3-1 under carbon and nitrogen starvation with the aim to reveal V-ATPase subunit C is involved in autophagy pathway.The following results are obtained:1.RT-PCR and GUS staining indicated that AtVHA-C is widely expressed in seedling,hypocotyl,leaf,flower and capsule in Arabidopsis thaliana.The result of transient expression in tobacco epidermal cells shown that AtVHA-C expressed on the membrane,microfilament and cell nucleus.2.Compared to the wild type,the mutant were more sensitive to nitrogen and carbon starvation.After nitrogen starvation,cell dead seriously and ROS accumulated in leaves of det3-1.After carbon starvation,all of det3-1 died.And ROS began accumulating when 4-week old det3-1 seedling were treated with 3 days darkness.3.Compared to the wild type,the formation and transport of autophagosomes were significantly affected in mutants.The results of Western Blot shown that the ATG8 protein in det3-1 increased at first and then decreased after nitrogen starvation.After nitrogen starvation 16 h,autophagosome formation in ATG8e-GFP/det3-1 was detected by Spin Disc,showing that the number of autophagosomes in the mature root zone of det3-1 was close to that in wild type.Howere,the number of autophagosomes in the mature root zone of det3-1 were less than in wild type after treatment with ConcA.After nitrogen starvation 0 d,1 d,2 d,we analysised the change of ATG8 eGFP fusion protein and free GFP protein by Western Blot.And fiound that free GFP remained essentially unchanged after nitrogen starvation.The number of autophagosome in mature root zone of det3-1 is 0.5 times that in the wild type after carbon starvation,but after the addition of ConcA,the number of autophagosome in the mature root zone of det3-1 is close to wild type.The variation trend of ATG8 protein in det3-1 is close to that of wild type after darkness treatment.4.The expression levels of autophagy related genes and potential autophagy pathway related genes in the mutant were significantly affected.Extracted the RNA of det3-1 after nitrogen starvation 0 d,2 d.qRT-PCR was performed after reverse transcription to detect the expression of autophagy related genes and some genes that may be related to autophagy pathway.The results indicated that nitrogen starvation in addition to affecting the expression of autophagy related gene ATG2,ATG8 a,ATG18a,the expression of ACS2,ACS8 and SAG113 were also changed,which are related to ethylene synthesis and ABA signal respectively.After treatment with exogenous ABA and ACC,shown that ABA can affect the cotyledon greening of det3-1,while ACC had no effect on germination,cotyledon greening and the root length of det3-1.To sum up,in this study we explored the effects of nitrogen starvation and carbon starvation on the levels of protein and transcription in det3-1 mutant by using the methods of cell biology and molecular biology,and identified that det3-1 is an autophagy defective mutant.It lays a foundation for further research on the involvement of V-ATPase subunit C in autophagy.And providing a new idea and theoretical basis for the exploration of autophagy mechanism and physiological function of V-ATPase.