Detecting autophagy in Arabidopsis roots by membrane-permeable cysteine protease inhibitor E-64d and endocytosis tracer FM4–64

Autophagy is the process by which cells degrade their own components in lysosomes or vacuoles. Autophagy in tobacco BY-2 cells cultured in sucrose-free medium takes place in formed, autolysosomes in the presence of a cysteine protease inhibitor. The autolysosomes in BY-2 cells are located in the endocytotic pathway and thus can be stained with fluorescent endocytosis marker FM4–64. In the present study, in order to detect autophagy in the root cells of Arabidopsis, we incubated root tips from Arabidopsis seedlings in culture medium containing the membrane-permeable cysteine protease inhibitor E-64d and FM4–64, and examined whether autolysosomes stained with FM4–64 are accumulated. The results suggest that autophagy accompanying the formation of autolysosomes also occurs in Arabidopsis root cells. Such autophagy appeared to occur constitutively in the root cells in nutrient-sufficient culture medium. Even in atg5 mutants in which an autophagy-related gene is disrupted, accumulation of the structures stained with FM4–64, which likely correspond to autolysosomes, was seen although at lower level than in wild type roots.     

AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells

In Arabidopsis root tips cultured in medium containing sufficient nutrients and the membrane-permeable protease inhibitor E-64d, parts of the cytoplasm accumulated in the vacuoles of the cells from the meristematic zone to the elongation zone. Also in barley root tips treated with E-64, parts of the cytoplasm accumulated in autolysosomes and pre-existing central vacuoles. These results suggest that vacuolar and/or lysosomal autophagy occurs constitutively in these regions of cells. 3-Methyladenine, an inhibitor of autophagy, inhibited the accumulation of such inclusions in Arabidopsis root tip cells. Such inclusions were also not observed in root tips prepared from Arabidopsis T-DNA mutants in which AtATG2 or AtATG5, an Arabidopsis homolog of yeast ATG genes essential for autophagy, is disrupted. In contrast, an atatg9 mutant, in which another homolog of ATG is disrupted, accumulated a significant number of vacuolar inclusions in the presence of E-64d. These results suggest that both AtAtg2 and AtAtg5 proteins are essential for autophagy whereas AtAtg9 protein contributes to, but is not essential for, autophagy in Arabidopsis root tip cells. Autophagy that is sensitive to 3-methyladenine and dependent on Atg proteins constitutively occurs in the root tip cells of Arabidopsis.     

Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.     

3-methyladenine inhibits autophagy in tobacco culture cells under sucrose starvation conditions

Tobacco (Nicotiana tabacum) culture cells perform autophagy and degrade cellular proteins in response to sucrose starvation. When protein degradation is blocked by the cysteine protease inhibitor E-64c, lysosomes containing particles of cytoplasm (autolysosomes) accumulate in the cells. Therefore, using light microscopy, we can determine whether cells have performed autophagy. In this study, we investigated whether or not 3-methyladenine (3-MA), which is a known inhibitor of autophagy in mammalian cells, blocks autophagy in tobacco culture cells. The accumulation of autolysosomes was blocked by the addition to the culture media of 5 mM 3-MA together with E-64c. We did not detect autolysosomes or structures thought to be involved with autophagy, such as autophagosomes, accumulating in these cells, as observed by electron microscopy. 3-MA blocked cellular protein degradation without any effect on cellular protease activity. In mammalian cells, phosphatidylinositol 3-kinase (PtdIns 3-kinase) is a putative target of 3-MA. The PtdIns 3-kinase inhibitors wortmannin and LY294002 also inhibited the accumulation of autolysosomes in tobacco culture cells. These results suggest that (1) 3-MA inhibits autophagy by blocking the formation of autophagosomes in tobacco culture cells, and (2) PtdIns 3-kinase is essential for autophagy in tobacco cells.     

Autophagy in tobacco BY-2 cells cultured under sucrose starvation conditions: isolation of the autolysosome and its characterization

Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where autolysosomes formed de novo act as the major lytic compartments. The digestion process in autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of autolysosomes. In the present study, we have investigated several properties of autolysosomes in tobacco cells. Electron microscopy showed that the autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H(+)-ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment-the autolysosome-also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.     

Constitutive autophagy in plant root cells

In previous studies, using a membrane-permeable protease inhibitor, E-64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy-related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient-sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose-free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient-limiting conditions but also in cell growth and root hair formation.     

Endosomal proteases facilitate the fusion of endosomes with vacuoles at the final step of the endocytotic pathway

The mechanism by which plasma membrane proteins are transported to vacuoles for degradation has not been well characterized in plants. To clarify how plasma membrane proteins are degraded, we monitored the endocytotic pathway in tobacco suspension-cultured BY-2 cells with a fluorescent endocytosis marker, FM4-64. Because of the efficient and rapid delivery of endosomes to the vacuoles, endosomes were scarcely detectable. Interestingly, we found that E-64d, an inhibitor of papain family proteases, caused the accumulation of a large number of endosomes in the cells under the sucrose-starved condition. This result indicates that E-64d attenuates the fusion of endosomes with vacuoles. We identified two papain homologues, which are localized in the endosomes, with a biotinylated inhibitor. We designated them as endosome-localized papains (ENPs). Immunofluorescent analysis revealed that vacuolar sorting receptor, a marker of prevacuolar compartment (PVC), was localized in the endosomes. This result and their acidic nature show that the endosomes correspond to PVC. These results suggest that ENPs facilitate the final step in the vacuolar trafficking pathway under the sucrose-starved condition. We further examined the effects of E-64d on two transgenic Arabidopsis plants that constitutively express a fusion protein composed of green fluorescent protein (GFP) and a plasma membrane protein (GFP-PIP2a or GFP-LTI6b). GFP fluorescence was observed on the plasma membrane of root cells in these transgenic plants. Treatment with E-64d induced the accumulation of GFP-fluorescent endosomes and inhibited the degradation of these fusion proteins. No GFP fluorescence was observed in vacuoles in E-64d-treated transgenic plants. Taken together, these results suggest that endosomal proteases are required for the fusion of endosomes with vacuoles at the final step in the endocytotic pathway for degradation of plasma membrane proteins in plants.     

Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates

Autophagy triggered by carbohydrate starvation was characterized at both biochemical and structural levels, with the aim to identify reliable and easily detectable marker(s) and to investigate the factors controlling this process. Incubation of suspension cells in sucrose- free culture medium triggered a marked degradation of the membrane polar lipids, including phospholipids and galactolipids. In contrast, the total amounts of sterols, which are mainly associated with plasmalemma and tonoplast membranes, remained constant. In particular, phosphatidylcholine decreased, whereas phosphodiesters including glycerylphosphorylcholine transiently increased, and phosphorylcholine (P-Cho) steadily accumulated. P-Cho exhibits a remarkable metabolic inertness and therefore can be used as a reliable biochemical marker reflecting the extent of plant cell autophagy. Indeed, whenever P-Cho accumulated, a massive regression of cytoplasm was noticed using EM. Double membrane-bounded vacuoles were formed in the peripheral cytoplasm during sucrose starvation and were eventually expelled into the central vacuole, which increased in volume and squeezed the thin layer of cytoplasm spared by autophagy. The biochemical marker P-Cho was used to investigate the factors controlling autophagy. P-Cho did not accumulate when sucrose was replaced by glycerol or by pyruvate as carbon sources. Both compounds entered the cells and sustained normal rates of respiration. No recycling back to the hexose phosphates was observed, and cells were rapidly depleted in sugars and hexose phosphates, without any sign of autophagy. On the contrary, when pyruvate (or glycerol) was removed from the culture medium, P-Cho accumulated without a lag phase, in correlation with the formation of autophagic vacuoles. These results strongly suggest that the supply of mitochondria with respiratory substrates, and not the decrease of sucrose and hexose phosphates, controls the induction of autophagy in plant cells starved in carbohydrates.     

Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been studied primarily using morphological analyses. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeast and plants. It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome. In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation. To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein-ATG8 fusion (GFP-ATG8). In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in a T-DNA insertion double mutant of the ATG4s (atg4a4b-1), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions. In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor. Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins. The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.     

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.     

Degradation of membrane phospholipids in plant cells cultured in sucrose-free medium

It has been generally accepted that autophagy contributes to the degradation of cellular components under nutrient starvation conditions. In a previous study, however, we showed that the degradation of membrane phospholipids occurs mainly by mechanisms distinct from autophagy in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells. In response to deprivation of sucrose, the amounts of total phospholipids and a major phospholipid, phosphatidylcholine (PC), decreased. 3-Methyladenine, which inhibits autophagy, did not affect the degradation of total phospholipids or PC. On the other hand, glycerol inhibited PC degradation although it did not block autophagy. In the present study, we labeled intracellular phospholipids by loading cells with a fluorochrome-labeled fatty acid and observed cellular morphology by fluorescence microscopy. Most cellular membrane structures were stained at the start of starvation; but 12 h after starvation treatment, concomitant with PC degradation, fluorescence on membranes disappeared and instead the central vacuole became fluorescent. 3-Methyladenine did not inhibit this process, whereas glycerol did. These results suggest that the degradation of membrane phospholipids can be traced by light microscopy and support the notion that autophagy is not a main contributor to the degradation of membrane phospholipids in tobacco cells cultured in sucrose-free medium.     

Constitutive Autophagy in Plant Root Cells

In previous studies, using a membrane-permeable protease inhibitor, E-64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy-related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient-sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose-free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient-limiting conditions but also in cell growth and root hair formation.

Addendum to:

AtATG Genes, Homologs of Yeast Autophagy Genes, are Involved in Constitutive Autophagy in Arabidopsis Root Tip Cells

Y. Inoue, T. Suzuki, M. Hattori, K. Yoshimoto, Y. Ohsumi and Y. Moriyasu

Plant Cell Physiol 2006; 47:1641-52     

Vacuole Fusion Regulated by Protein Phosphatase 2C in Fission Yeast

The gene ptc4+ encodes one of four type 2C protein phosphatases (PP2C) in the fission yeast Schizosaccharomyces pombe. Deletion of ptc4+ is not lethal; however, Deltaptc4 cells grow slowly in defined minimal medium and undergo premature growth arrest in response to nitrogen starvation. Interestingly, Deltaptc4 cells are unable to fuse vacuoles in response to hypotonic stress or nutrient starvation. Conversely, Ptc4 overexpression appears to induce vacuole fusion. These findings reveal a hitherto unrecognized function of type 2C protein phosphatases: regulation of vacuole fusion. Ptc4 localizes in vacuole membranes, which suggests that Ptc4 regulates vacuole fusion by dephosphorylation of one or more proteins in the vacuole membrane. Vacuole function is required for the process of autophagy that is induced by nutrient starvation; thus, the vacuole defect of Deltaptc4 cells might explain why these cells undergo premature growth arrest in response to nitrogen starvation.     

Protein Aggregates are Transported to Vacuoles by Macroautophagic Mechanism in Nutrient-Starved Plant Cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated with nutrient limit medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit authophagic processes, inhibited the transport of RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP–NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to otherorganelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation occurs in plant cells.     

Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated in nutrient limited medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit autophagic processes, blocked the transport of the RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP-NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to other organelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation can occur in plant cells.     

Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver

We isolated membranes from leupeptin-induced autophagic vacuoles and compared them with lysosomal membranes purified from dextran-administered rats. In protein composition, autophagic vacuole membranes prepared from long term-starved (36 h) rats bear marked resemblance to lysosomal membranes, whereas vacuole membranes prepared from short term-starved (12 h) animals differ significantly from lysosomal membranes. Immunoblotting analyses showed that only autophagic vacuole membranes from short term-starved rats possess endoplasmic reticulum markers such as cytochrome P450 and NADPH-cytochrome c reductase. None of the membranes contain sialyltransferase, a Golgi membrane marker. In experiments in which rats were starved after feeding to induce autophagy, the appearance of the endoplasmic reticulum markers occurred during 6-12 h of starvation, concomitantly with increases in vacuolar proteins and sequestered cytosolic aldolase. The endoplasmic reticulum membrane markers and sequestered aldolase declined gradually after 20-36 h of starvation, suggesting that prolonged starvation causes no further increase in the formation of autophagic vacuoles but an increase in the population of matured autophagic vacuoles. Thus, the prominent markers of endoplasmic reticulum from which autophagosomes originate are well preserved in autophagic vacuole membranes, and retention of these markers is highly dependent on the formation and subsequent maturation process of autophagic vacuoles.     

Contribution of the plasma membrane and central vacuole in the formation of autolysosomes in cultured tobacco cells

Autolysosomes accumulate in tobacco cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We characterized these plant autolysosomes using fluorescent dyes and green fluorescent protein (GFP). Observation using the endocytosis markers, FM4-64 and Lucifer Yellow CH, suggested that there is a membrane flow from the plasma membrane to autolysosomes. Using these dyes as well as GFP-AtVam3p, sporamin-GFP and gamma-VM23-GFP fusion proteins as markers of the central vacuole, we found transport of components of the central vacuole to autolysosomes. Thus endocytosis and the supply from the central vacuole may contribute to the formation of autolysosomes.     

Autophagy is not a main contributor to the degradation of phospholipids in tobacco cells cultured under sucrose starvation conditions

Net degradation of cellular components occurs in plant cells cultured under starvation conditions, and autophagy contributes to the degradation of intracellular proteins. In this study, we investigated the degradation of membrane phospholipids by autophagy in cultured tobacco (Nicotiana tabacum) cells. The amounts of total phospholipids and a major phospholipid, phosphatidylcholine (PC), decreased, whereas phosphorylcholine, a degradation product of PC, increased in response to deprivation of sucrose. The addition of glycerol to the culture medium inhibited both the degradation of phospholipids and the concomitant increase of phosphorylcholine. Glycerol, however, did not block autophagy, which was assessed by the accumulation of autolysosomes in the presence of a cysteine protease inhibitor. On the other hand, 3-methyladenine, an inhibitor of autophagy, did not affect the net degradation of PC. We labeled intracellular phospholipids by loading cells with a fluorochrome-labeled fatty acid and chased it under sucrose-free conditions. Glycerol slowed down the decrease in the amount of fluorochrome-labeled PC, suggesting that it inhibits the degradation process of PC. These results show that phospholipids are degraded by mechanisms different from autophagy in tobacco cells cultured under sucrose-free conditions.     

Simplification of vacuole structure during plant cell death triggered by culture filtrates of Erwinia carotovora

Vacuoles are suggested to play crucial roles in plant defense-related cel death. During programmed cel death, previous live cel imaging studies have observed vacuoles to become simpler in structure and...