? In the early identification of autophagy genes in tobacco, the author found that the expression of ATGs related to autophagy increased greatly during pollen germination and reached the maximum in mature pollen, suggesting that autophagy may participate in the normal growth and development of plants. So the author wants to use tobacco as a model to explore whether this is the case.
? Fortunately, the author found that autophagy activity was greatly enhanced during pollen germination by various means. 30 minutes after germination, autophagy was observed by transmission electron microscope. When Con A (concanavalin A, which can acidify vacuoles and prevent autophagy from degrading) is used, autophagy is found to be enriched in vacuoles near germination holes. Immunoanalysis of autophagy labeled with ATG8 specific antibody under optical microscope showed that autophagy had obvious point aggregation at germination hole. RT-QCPR also showed that most autophagy-related genes were up-regulated at 1 hour after germination, and then down-regulated, suggesting that autophagy was involved in pollen germination.
? In order to prove that autophagy is really involved in the process of pollen germination, kill him. So they used autophagy inhibitor 3-MA, that is, phosphatidylinositol 3- kinase (PtdIns3K) inhibitor to treat pollen germination in vitro. It was found that the pollen treated with inhibitors did not germinate much. Moreover, the inhibitor treatment of germination has dose effect (qualitative experiment). Subsequently, autophagy was labeled with ATG8 antibody, and it was found that the formation of autophagy was indeed reduced (about 70%) after adding 3-MA (quantitative experiment). So far, it can be said that autophagy does participate in the process of pollen germination.
? In order to further explore the role of autophagy in pollen germination, the author compared normal pollen germination with 3-MA treatment. It was found that in the process of 3-MA treatment, the attachment of germination holes would form a convex cytoplasm structure, which was obviously different from other cytoplasm. The author thinks that autophagy is mainly to remove this prominent cytoplasmic structure.
? In previous studies, it was known that autophagy activity was related to ATG family genes, so the author began to explore its mechanism. So the author screened three single copies of ATG2, ATG5 and ATG7 genes (not PtdIns3K complex) and constructed RNAi vectors respectively. It was found that the down-regulation of ATG2, ATG5 and ATG7 (in the annex) did cause the decline of pollen germination rate, which was similar to the phenotype of 3-MA treatment. Moreover, the down-regulation of ATGs will not lead to the decline of pollen vitality, but only about 30%-50% of pollen can germinate.
? The focus of the next work is undoubtedly to explore the relationship between pollen germination failure caused by ATGs and autophagy. By using ATG8 and ATG 13 as the reporter genes of Western blot, we can see that the expression levels of ATG8 and ATG 13 in RNAi plants are down-regulated, suggesting that when one of the genes is down-regulated, the autophagy level will also be down-regulated. Immunofluorescence detection showed that the number of autophagy in RNAi plants decreased significantly. In order to distinguish whether the decrease of autophagy leads to decrease or rapid degradation, Con A can't restore the number of autophagy to the wild-type level, which indicates that ATGs is involved in the formation of autophagy. Use crispr mutant atg5. It further shows that there are indeed problems in the formation of autophagy.
? In order to further explore the relationship between the formation of autophagy and ATG, the author analyzed the obesity degree of ATG8, because it is very important for the formation of autophagy. All membrane components were collected from the pollen of WT and ATGs-RNAi, and then the lipidation of ATG8 was determined by protein blot analysis before or after PLD (phospholipase D) pretreatment. After SDS-PAGE, the migration band of at G8–PE is usually detected in western blot, which is faster than that of ATG8. It can be seen that the wild type runs faster after treatment, which proves that they are lipidated in G8. However, in ATGs-RNAi plants, the lipid level of ATG decreased to a general level. In ATGs-RNAi plants, it was also found that there was a raised cytoplasm layer at the germination hole, and its area was quantified.
In order to explore the relationship between autophagy and lipid in the early stage of pollen germination, the pollen of wild-type and ATG RNAi plants was analyzed by liquid chromatography-mass spectrometry. Several sphingolipids, phospholipids and glycerides were identified and quantified in wild-type and ATG silent pollen. Orthogonal projection-discriminant analysis (OPLS-DA) of latent structure showed that there was obvious separation between wild-type and ATG_RNAi strains, indicating that there were obvious differences in lipid composition among ATG _ RNAi strains. Then, the lipids involved in autophagy were analyzed in detail. Phosphatidylinositol (PI), phosphatidylethanolamine (PE) and phosphatidylcholine are involved in the formation of autophagy, among which PI can be phosphorylated by VPS34 to form PtdIns3K during autophagy nucleation, and the expression level in RNAi plants is greatly reduced. In addition, in the pollen of ATGs_RNAi, the expression levels of PE and phosphatidylcholine, which are necessary for vesicle nucleation and phagocyte expansion, respectively, also decreased, accompanied by the increase of some lysosomes and lysophosphatidylcholine.
In addition, in mammals, exogenous ceramide (Cer) of sphingolipids can activate autophagy by inhibiting amino acid uptake and inducing the expression of BECN 1/Beclin 1, that is, mammalian orthologs of yeast VPS30/ATG6. Interestingly, the level of Cer in ATGs_RNAi pollen was significantly higher than that in wild type. The redistribution of phospholipid cardiolipin (CL) from the inside of mitochondria to the outside of mitochondria is very important for autophagy of damaged mitochondria. Compared with wild type, the accumulation level of chlorine in RNAi plant pollen is higher. Phosphatidylglycerol (an important substrate for CL synthesis) is also accumulated in the pollen of ATGs_RNAi. Similarly, compared with WT, the level of lysophosphatidylglycerol (the precursor of phosphatidylglycerol synthesis) in the pollen of ATGs_RNAi also increased. Triacylglycerol (TAG) is the main component stored in liposomes in many plant pollen grains. Inhibition of autophagy may lead to the increase of lipids in plants and mammals. Therefore, the author focused on the composition of TAG and diacylglycerol (DAG, an intermediate metabolite in TAG synthesis). A lot of accumulation of TAG and DAG was observed in ATGs_RNAi. Interestingly, a new amphipathic lipid subclass, ω-hydroxy fatty acids, also accumulated high levels in autophagy-deficient pollen. The above research shows that the change of membrane composition caused by autophagy ensures the normal germination of pollen.
Finally, the author used tandem mass spectrometry (TMT) for quantitative mass spectrometry analysis, and discussed the changes of downstream protein expression of ATG2 and ATG5. Among them, 4.3% and 2.8% were up-regulated in ATG2- and ATG 5-silent poll, and 2.6% and 2.8% were down-regulated. Therefore, the authors believe that autophagy pathway plays an important role in pollen protein formation. Moreover, there are many overlaps between up and down in ATG2_RNAi and ATG5_RNAi, which may regulate the same pathway. However, GO analysis showed that ATG2 mainly enriched the components related to ribosomal protein and ribonucleoprotein, while ATG5 enriched the components related to peptidase and proteasome, indicating their functional differences. In terms of cell composition, we can find that the protein related to mitochondrial proton transport ATP synthase complex is obviously down-regulated, and the cell membrane composition is obviously up-regulated. In biological processes, down-regulated proteins are enriched in the biosynthesis of cellular nitrogen compounds and the coupling of ATP synthesis and proton transport, while up-regulated proteins are enriched in signal transduction mediated by small GTpase. Therefore, the author thinks that ATG2 and ATG5 mainly affect pollen germination through the synthesis of mitochondrial ATP and protein. In order to confirm this result, the author also identified the enrichment pathway, and found that both up-regulated and down-regulated proteins were classified as pathways related to oxidative phosphorylation.
This paper reveals how autophagy participates in the process of pollen germination and its influence on lipid components and downstream proteins. However, these ATG proteins seem to play a role in pollen germination, which seems redundant, but every RNAi plant has a phenotype, which makes me feel very strange. It would be better if we could do double mutation or triple mutation. In addition, what is the function of another ATG whose expression changes during pollen germination? Need further excavation.