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Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2009, Article ID 451357, 14 pages doi:10.1155/2009/451357 Review Article Ger ´ aldine Mitou, Hikmet Budak, and Devrim Gozuacik Biological Science and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Orhanli, Tuzla 34956, Istanbul, Turkey Correspondence should be addressed to Devrim Gozuacik, email@example.com Received 7 December 2008; Revised 15 May 2009; Accepted 18 June 2009 Recommended by Boulos Chalhoub Autophagy (or self eating), a cellular recycling mechanism, became the center of interest and subject of intensive research in recent years. Development of new molecular techniques allowed the study of this biological phenomenon in various model organisms ranging from yeast to plants and mammals. Accumulating data provide evidence that autophagy is involved in a spectrum of biological mechanisms including plant growth, development, response to stress, and defense against pathogens. In this review, we brieﬂy summarize general and plant-related autophagy studies, and explain techniques commonly used to study autophagy. We also try to extrapolate how autophagy techniques used in other organisms may be adapted to plant studies. Copyright © 2009 Ger ´ aldine Mitou et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction the cell to economize its resources, eliminate old/damaged organelles, and survive nutrient and other types of stress. Autophagy, literally meaning self (auto) eating (phagy), is For example, in plants under conditions causing cellular an evolutionarily conserved and highly regulated catabolic and organismal stress such as starvation, drought, and other abiotic stress, autophagy is upregulated [5–8]. Autophagy is process that leads to the degradation of cellular components using lysosomal/vacuolar degradation machinery of the also involved in physiological phenomena including plant same cell. Depending on the mechanism of transport to development, senescence, and immune response [9–11]. In some cases, autophagy can function as a nonapoptotic and lysososome/vacuole, at least three forms of autophagy have been described: “Macroautophagy” is characterized by the alternative programmed cell death mechanism, and its role in plant cell death was explored [12–15]. As a consequence engulfment of long-lived proteins and organelles in de novo formed double-/multimembrane vesicles called autophago- of its involvement in several important physiological and somes or autophagic vesicles. These vesicles subsequently pathological phenomena, autophagy became one of the fastest expanding ﬁelds of molecular biology in recent years. deliver their cargo to the lysosome or vacuole for degrada- tion. In another form of autophagy, called “microautophagy,” In this review, we will brieﬂy summarize the mechanisms lysosome/vacuole directly engulfs cytosolic components of autophagy in general and particularly plant autophagy, list commonly used techniques to detect and quantify through an invagination of its membrane [1, 2]. A third common form of autophagy is called “chaperone-mediated autophagy, and ﬁnally discuss their utility in plant autophagy autophagy” (CMA). CMA is a very selective process during detection. An exhaustive summary of the autophagy mecha- nisms is beyond the scope of this review. The readers may which proteins with a KFERQ consensus peptide sequence are recognized by a chaperone/cochaperone complex and ﬁnd an in-depth discussion of the mechanistic aspects of autophagy in recently published reviews [5, 9, 16]. delivered to the lytic compartment in an unfolded state [3, 4]. Macroautophagy is the most studied form of autophagy. Macroautophagy (“autophagy” hereafter) occurs at basal 2. General Autophagy Mechanisms levels in growing cells, allowing them to recycle long-lived proteins and organelles . The cargo is degraded into So far, nearly 30 autophagy-related genes (depicted by the its building blocks (i.e., proteins to amino acids), helping acronym ATG) were identiﬁed using yeast mutants . 2 International Journal of Plant Genomics 6) Recycling Autolysosome Autophagosome 1) Induction 2) Nucleation 3) Vesicle expansion 4) AV/lysosome and completion 5) Degradation fusion (a) Vacuole Autophagosome Induction (b) “Autolysosome-like structure” Autophagosome Vacuole Induction (c) Figure 1: Autophagy mechanism and alternative pathways for autophagosomes in plants. (a) Following an upstream stimulus, such as starvation, double membrane vesicles, autophagosomes, appear and engulf portions of cytosol, long-lived proteins, and organelles such as mitochondria. Autophagosomes eventually fuse with lysosomes, endosomes, or vacuole. Autophagosomes are degraded together with their cargo and the building blocks are pumped back into the cytosol for reuse. (b) Autophagosomes may fuse directly with the vacuole (observed in A. thaliana) (c) or, may ﬁrst transform into lysosome-like acidic and lytic structures and, fusion with the central vacuole may occur as a secondary event (observed in tobacco plant). Plant and mammalian orthologues of most of these genes ATG13)[25, 26]. Second mechanism is related to the mod- and proteins are now characterized. Data obtained from iﬁcation by Tor of an autophagy protein complex containing these studies underline the fact that the basic machinery Atg1 and Atg13. Active Tor induces hyperphosphorylation of autophagy is preserved from yeast to higher eukaryotes. of Atg13 inhibiting its association with Atg1 (AtAtg1 in A. Autophagy proceeds through ﬁve distinct phases: namely, thaliana and ULK1 (Unc-51-like kinase1) in mammals), a induction, nucleation, vesicle expansion and completion, serine/threonine kinase required for autophagy . Tor autophagosome/lysosome fusion, and cargo degradation [9, inactivation leads to rapid dephosphorylation of Atg13 and 18](Figure 1(a)). an increase in the aﬃnity of this protein for Atg1. Atg1-Atg13 association induces autophosphorylation and activation of Atg1, promoting autophagy [27–30]. Recent evidences indi- cate that Atg1-13 complex regulates recycling of Atg proteins 2.1. Induction. This is the phase where upstream signaling such as Atg9 and Atg23 functioning at the autophagy mechanisms leading to autophagy activation are turned on. organization site called PAS (for the preautophagosomal Many of these pathways are integrated by the “Target of structure) . rapamycin (Tor)” protein [19–21]. Tor is a serine/threonine kinase regulated in response to variation in amino acids, ATP, and growth factors. Downregulation of Tor activity 2.2. Nucleation. While the origin of the lipid donor mem- correlates with autophagy stimulation . Tor pathway branes in autophagy is still obscure, endoplasmic reticulum, and its eﬀect on autophagy were preserved in plants. Yet, Golgi, and a so far undetermined organelle called “the structural diﬀerences exist between Tor proteins in plants phagophore” were suggested as lipid providers to autophago- and other eukaryotes, therefore, rapamycin, a widely used somes. Whatever is the origin, autophagosomal membranes speciﬁc inhibitor of Tor, cannot be used to study autophagy are build up de novo as crescent-shaped structures in in plants [23, 24]. PAS. In yeast, PAS is a prominent structure next to the Tor inactivation induces autophagy at least by two mech- vacuole, but in higher eukaryotes, several sites are involved. anisms in yeast. The ﬁrst involves activation of transcription Nucleation of autophagosomes is initiated by a protein factors called GLN3 (nitrogen regulatory protein) and GCN4 complex including Vps34, a class III phosphatidylinositol 3- (General Control Nondepressible), leading to transcriptional OH kinase (PI3K), and Atg6/Vps30 (Beclin1 in mammals). upregulation of some of the ATG genes (e.g., ATG1 and Together with other regulatory proteins such as UVRAG (UV International Journal of Plant Genomics 3 radiation Resistance Associated Gene), Bif-1, and Ambra, 3. Plant Autophagy Atg6-containing complex plays a role in the regulation Both microautophagy and macroautophagy are functional in of Vps34 activity. PI3K activity of Vps34 leads to the plants . Mechanisms of these pathways are similar to those accumulation of phosphatidylinositol 3-phosphate (PI3P). described in other model organisms. PI3P produced by Vps34 serves as a landing pad on PAS for In plant microautophagy, the target material is directly proteins involved in autophagosome formation such as Atg18 engulfed by an invagination of the tonoplast. Cargo- and Atg2 [16, 32, 33]. containing vesicle pinches oﬀ to be released inside the vacuole and degraded within the lumen. Microautophagy 2.3. Vesicle Expansion and Completion. Two ubiquitination- was involved in accumulation of storage proteins, lipids, and like conjugation systems play a role in autophagosome bio- degradation of starch granules in developing plants [49, 50]. genesis. In the ﬁrst reaction, Atg12 is conjugated to Atg5 in a As in other organisms, the macroautophagy (hereafter covalent manner . The conjugation reaction starts with “autophagy”) in plants is a process that starts with the the activation of Atg12 by an ubiquitin-activating enzyme formation of cup-shaped membranes in the cytoplasm. After (E1)-like protein Atg7. Atg12 is then transferred to Atg10, completion, autophagosomes have at least two destinations an ubiquitin-conjugating-like enzyme (E2)-like protein [35, in plants. They may fuse with the tonoplast and be directly 36]. Finally, Atg12 is covalently conjugated to Atg5. The delivered to the lumen of the vacuole as seen in Arabidopsis. conjugation allows the formation and stabilization of a Alternatively, autophagosomes may ﬁrst transform into larger complex containing Atg12, Atg5, and Atg16 . This lysosome-like acidic and lytic structures and, fusion with the protein complex is necessary for the second ubiquitination- central vacuole may occur as a secondary event (Figures 1(b) like reaction to occur and to allow autophagosome mem- and 1(c))[51, 52]. brane elongation. Atg12/5/16 complex localizes to the outer In the model plant Arabidopsis thaliana, 25 orthologs of membrane of the forming autophagosome, and, dissociates 12 yeast ATG genes were identiﬁed [44, 53–55]. Some exist from it as soon as the vesicle is completed, underlining the as a single copy (i.e., Atg3 and Atg5) and others as multiple fact that its role is regulatory rather than structural . copies (i.e., Atg1 and Atg8). Functional domains of these The second ubiquitination-like reaction involves Atg8 Arabidopsis proteins were well conserved during evolution, protein (microtubule-associated protein light chain-3 or indicating preservation of basic autophagy mechanisms in shortly LC3 in mammals). E1-like protein Atg7 activates Atg8 plants. Indeed, complementation tests in ATG mutant yeast and transfers it to Atg3. While Atg7 is common to both con- strains using some of the plant Atg proteins conﬁrmed jugation reactions, E2-like protein Atg3 is speciﬁc for Atg8 the preservation of their function . Moreover, gene conjugation to a lipid molecule (phosphatidylethanolamine, targeting studies in whole plants demonstrated that plant PE) . Prior to conjugation, Atg8 has to be cleaved at its genes of all tested autophagy proteins (i.e., for Atg7, Atg9 and carboxy-terminus by Atg4, allowing the access of the lipid Atg5-Atg12) were necessary for autophagosome formation molecule to a Glycine residue on Atg8. Lipidation reaction following various types of stress [44, 53, 55]. Furthermore, is reversible since Atg4 can also cleave the conjugated lipid, some ATG genes were upregulated under stress conditions enabling recycling of Atg8. Recent data provide evidence that stimulating autophagy [7, 56–61]. A list of Atg genes together with Atg3, Atg12/5 complex is directly responsible identiﬁed in Arabidopsis and the phenotypes caused by their for Atg8-PE conjugation . The yeast Atg8 has several modiﬁcation are depicted in Table 1. orthologues and isoforms in plants [41–43]. In the model plant Arabidopsis thaliana, at least 9 Atg8 proteins were 3.1. Basal Autophagy in Plants. Autophagy is constitutively described . active in plant cells as in other organisms. Indeed, incubation of root tips with vacuolar enzyme inhibitors led to the 2.4. Autophagosome/Lysosome Fusion and Degradation. accumulation of autophagic vesicles as autolysosome-like Autophagosomes fuse with late endosomes or lysosomes to structures and in the vacuole. When cysteine protease form autolysosomes. Speciﬁc factors have been implicated inhibitor, E64d, was used to inhibit autophagy, autophagic in this step. A Vps complex and Rab GTPases proteins vesicles accumulated inside vacuoles in Arabidopsis cells . are involved in the organization of the fusion site. Then, Similarly, growth of tobacco cells in the presence of E64d SNAREs proteins (SNAP as soluble NSF attachment protein led to the accumulation of autolysosome-like structures receptor)  form a complex which serves as a bridge outside the vacuole . Autophagy-speciﬁc inhibitor 3- between the two organelles [46, 47]. MA blocked the accumulation of autophagosomes and autolysosomes, demonstrating that autophagy is responsible 2.5. Recycling. In the lumen of lysosome/vacuole, lipases for vesicle accumulation [52, 62]. Expression of a GFP fusion such as Atg15 ﬁrst degrade the remaining autophagic construct of Atg8f (an autophagy marker in Arabidopsis) membrane and the cargo is then catabolized by lysosomal resulted in the accumulation of this marker protein in the lytic enzymes . Following the degradation of the vesicle, vacuole lumen. Atg8f accumulation was also detected in building blocks are carried to cytosol for further use. the presence of concanamycin A (a Vacuolar H(+)-ATPase Specialized lysosome membrane proteins play a role in this inhibitor blocking vacuolar degradation) . process including lysosomal-associated membrane proteins The role of constitutive autophagy in the degradation LAMP-1 and LAMP-2. of damaged or oxidized molecules was conﬁrmed using 4 International Journal of Plant Genomics mutants of AtAtg18a. These mutants produced greater grammed cell death (HR-PCD). The innate immunity is amounts of oxidized proteins and lipids in comparison to achieved through limitation of the infection with the death wild-type plants. Increased amount of oxidized protein and of cells surrounding the infected area . Studies using lipid generation in Atg18a-silenced plants underlined impor- autophagy gene mutant plants showed that an autophagy tance of autophagy for the degradation of oxidized molecules defect is associated with a failure to contain cell death at in plant cells [8, 63]. Therefore, as in other organisms, plant the infection site, leading to its spread into uninfected tissue basal autophagy seems to function to eliminate damaged [79–81]. Therefore, paradoxically, autophagy also plays a role organelles (e.g., chloroplast, a source of reactive oxygen in limiting cell death initiated during plant innate immune species in plants) and to clear damaged/abnormal proteins responses. Indeed, as seen in plants, autophagy is involved that accumulate in the cytoplasm . both in cell survival and cell death in various other organisms . 3.2. Autophagy in Plant Development. Theroleofautophagy for plant development was studied using several autophagy 4. Techniques to Study Autophagy gene mutants. Under nutrient-rich conditions, autophagy- Various techniques and tools were used to monitor and defective plants achieve normal embryonic development, evaluate autophagy. While transmission electron microscopy germination, shoot and root growth, ﬂower development, (TEM) analysis remains “the golden standard,” with the and seed generation [44, 53, 54]. When these plants are recent advances in the ﬁeld, several new molecular tools grown under carbon- or nitrogen-deﬁcient conditions, accel- are being introduced. The possibility of their usage in plant erated bolting, increased chlorosis, dark-induced senescence, autophagy research will be discussed. and a decrease in seed yield were observed. Therefore, autophagy seems to be a major mechanism of nutrient mobilization under starvation conditions in plants. 4.1. Electron Microscopy. Transmission electron microscopy (TEM) is one of the earliest tools used to characterize Autophagy plays a role during vacuole biogenesis as well. autophagy , and it is still one of the most reliable In arecentstudy,Yanoetal.  proposed that formation of vacuoles from tobacco BY-2 protoplasts involved an methods to monitor autophagy in cells and tissues. Yet, inter- pretation of the TEM data requires special expertise and there autophagy-like process. However, this process could not be inhibited by classical autophagy inhibitors such as 3-MA are several criteria to describe autophagosomes and autolyso- somes with precision. The hallmark of autophagosomes and wortmannin, suggesting that autophagy during vacuole is their double or multimembrane structures containing formation diﬀers from constitutive autophagy taking place under normal conditions or autophagy induced by stress. electron dense material with a density similar to that of the cytoplasm. Presence in autophagosomes of organelles such as mitochondria, chloroplasts, and endoplasmic reticulum 3.3. Autophagy, Stress, and Cell Death. When organisms including plants are exposed to adverse environmental (ER) strengthens the conclusion (Figure 2(b)). Autolyso- somes contain darker, degenerated, or degraded material and conditions, they develop responses to cope with stress and to survive. One of the major processes exploited by plant some of them are reminiscent of lysosomes/vacuole. Other cytoplasmic ﬁgures may be erroneously described cells for this purpose is autophagy. Stress conditions inducing as autophagosomes and autolysosomes. Degenerated mito- autophagy include sucrose, nitrogen, and carbon starvation, as well as oxidative stress and pathogen infection [8, 62, 66, chondria, folds of ER, or nuclear membrane may be mis- taken for autophagosomes [83–85]. Sometimes the typical 67]. For example, sucrose starvation has been reported to double membrane structure of autophagosomes may be dis- induce autophagy in rice , sycamore , and tobacco- cultured cells , and carbon starvation induced autophagy rupted (e.g., following infection with some pathogens) . Therefore, unbiased and clear identiﬁcation of autophago- in maize plants . Furthermore, autophagy participates somes using TEM requires extreme precaution. Combi- in the formation of protein storage vacuoles in seeds and nation of electron microscopy with immunogold-labelling cereal grains [71, 72], prolamin internalization to vacuole of autophagosome-speciﬁc markers such as Atg8/LC3 may in wheat , biogenesis of vegetative vacuoles in mature meristematic cells [74, 75], and degradation of proteins in allow a more objective and reliable interpretation depending on the experimental needs . Transmission electron protein storage vacuoles in mung bean [49, 76]. microscopy was successfully used to detect autophagy in Since plants have a rigid cell wall and they lack typical caspase proteases, apoptosis is not the mechanism utilized plants [61, 79]. by plants to degrade cellular components before cell death. During programmed cell death (PCD) in plants, vacuole and 4.2. Molecular Markers. Proteins that are involved in the cell size increase, organelles are taken up by vacuole and autophagy process or that are degraded speciﬁcally through subsequently degraded, and ﬁnally vacuole lyses resulting in autophagy have been used to monitor autophagic activity. cell death. These events overlap with the major character- Several of them are already in use in plants. Plants knock- istics of autophagy in plants [15, 77]. In the light of these out and transgenic for these markers are useful tools to study observations, the role of autophagy in plant programmed cell autophagy-related phenotypes under diﬀerent experimental death needs to be further investigated. conditions (see Table 1). Molecular techniques, such as To avoid spread of infection, plants developed an innate Atg8/LC3 dot formation, were successfully used for high- immune response, called the hypersensitive response pro- throughput screens of autophagy in various systems . International Journal of Plant Genomics 5 Table 1: Phenotypes caused by ATG gene modiﬁcations in Arabidopsis thaliana. E64d, inhibitor of lysosomal/vacuolar hydrolases; Concanamycin A, inhibitor of vacuolar (V-type) ATPase, preventing lysosomal/vacuolar degradation:HR-PCD (hypersensitive response programmed cell death). Genotype Reference(s) Phenotype Atg2-deﬁcient  No autophagic inclusions in root tips upon E64d treatment. Upon nitrogen starvation, no autophagosome formation and no delivery of Atg4a-/ Atg4b-deﬁcient  GFP-Atg8 to the vacuole.  Inhibition of rubisco containing body formation.  No autophagic vesicles in root tips after E64d treatment. Atg5-deﬁcient No formation of Atg5/12 complex. Defective in autophagy induced by  concanamycin A treatment. Senescence upon light and carbon or nitrogen limitation.   Male sterility. Atg6-deﬁcient  HR-PCD sensitive. Early senescence. Developmental defects and impaired pollen germination.  Atg7-deﬁcient  Hypersensitive to nutrient-limitation. Senescence. Atg8-transgenic [57, 66] Expression induced by starvation. Stress leads to premature aging.  Under carbon and nitrogen starvation, accelerated chlorosis. Atg9-deﬁcient Seed germination impaired and leaf senescence accelerated. Weak decrease of autophagic vesicle accumulation following E64d treatment.   Hypersensitive to nitrogen and carbon starvation. Early senescence and PCD. Atg10-deﬁcient No formation of Atg5/12 complex. Defective in autophagy induced by  concanamycin A treatment. Atg18a-transgenic  Hypersensitivity to sucrose and nitrogen starvation. Premature senescence. 4.2.1. Atg8/LC3 Dot Formation and Accumulation of Its following the usage of autophagy inhibitors. This method is Lipidated Form. Atg8/LC3 is covalently conjugated to a lipid a good quantitative tool to monitor activity in living cells molecule as a result of an ubiquitination-like reaction and, its by FACscan/ﬂow cytometer [92–94], especially using cells lipidation is required for autophagic membrane elongation derived from Atg8 transgenic plants. (see Section 2.3). In plants, several isoforms of Atg8/LC3 Nevertheless some precautions must be taken even when seem to be functional during autophagy mechanisms . using this popular molecular marker. Free Atg8 (or LC3-I) to During autophagy, Atg8/LC3 lipidation and recruitment to Atg8-PE (or LC3-II) ratio diﬀers among tissues, depending autophagic membranes changes its localization from diﬀuse on stimuli and antibodies that are used, therefore, reliable cytosolic to punctuate (Figure 2)[51, 54, 89, 90]. Moreover, controls must be added . To avoid misinterpretations in SDS-PAGE protein gels, the molecular weight of Atg8/LC3 due to kinetics of autophagy, it is highly advised to check changes from 18kDa (free cytosolic form, free Atg8, or LC3- Atg8/LC3 lipidation at several time points after signal I) to 16kDa (lipidated form, Atg8-PE (or LC3-II)) [41, application rather than using only one point in time . 54, 57]. Soon after the discovery of its autophagy-related The use of vacuolar/lysosomal degradation inhibitors will lipidation, Atg8/LC3 had become one of the main tools to help to conﬁrm that accumulation of the lipidated form is monitor autophagy. The localization change of an Atg8/LC3- indeed due to the canonical autophagy pathway. ﬂuorescent protein fusion construct (such as GFP-Atg8/LC3) Atg8/LC3 lipidation and cytosolic dot formation may is commonly used to detect autophagy in cells (Figure 2(a)) not always reﬂect activation of autophagy. It has been and in whole organisms including transgenic Arabidopsis and reported that high level GFP-Atg8/LC3 expression may also tobacco plants [38, 51, 54, 55, 57]. lead to dot formation even in nonautophagic cells  When working with isolated cells, quantiﬁcation of and in autophagy mutants . Furthermore, Atg8/LC3 GFP-Atg8/LC3 signal using FACscan/ﬂow cytometer may was found to associate with protein aggregates marked be used as an autophagy evaluation tool . In this with p62/SQSTM1 (see Section 4.2.7) in an autophagy- system, induction of autophagy led to a decrease in GFP- independent manner . Importantly, Atg8/LC3 lipidation Atg8/LC3 signal. Conversely the ﬂuorescent signal increased reﬂects an early stage in autophagosome formation and it 6 International Journal of Plant Genomics Control Starved (a) (b) Figure 2: GFP-Atg8/LC3 dot accumulation and TEM method to detect autophagic activity. (a) LC3 dot formation upon starvation in ﬁbroblasts isolated from GFP-Atg8/LC3 transgenic mice. The green dots are autophagic vesicles labelled by GFP-Atg8/LC3. (b) Transmission electron microscopic picture of an autophagic vesicle (arrow) in kidney of tunicamycin injected mouse. Note that in addition to cytoplasmic material, a mitochondrium (arrowhead) is also engulfed inside the double membrane vesicle. cannot be interpreted as autophagic activity per se [99, 100]. . WIPI-1 is a WD (Tryptophan and aspartic acid) Hence, this method should not be used as the only technique repeat protein  and as such, it may interact with to monitor autophagy and it has to be complemented PI3P and accumulate in dot-like structures (upon autophagy with other autophagy detection techniques including TEM induction by amino acid starvation other stimuli). WIPI-1 analysis . dots were shown to colocalize with Atg8/LC3 [107, 109]in human cells lines. Whether plant Atg18 protein might be 4.2.2. Atg6 and Phosphatidyl Inositol 3-Phosphate Detec- used as an autophagy marker has to be tested as homologues tion. The role of Atg6 in autophagy has been extensively are found in plants such as Arabidopsis. studied. As stated before, Atg6 regulates Vps34 class III phosphoinositide-3 kinase (PI3K) complex producing PI3P 4.2.5. Atg4 Activity. Cleavage of Atg8/LC3 by Atg4 cysteine that is involved in autophagic vesicle nucleation. Similar to protease is a crucial step before its lipidation. Recently, Atg8/LC3, intracellular localization change of a ﬂuorescent monitoring Atg8/LC3 cleavage by Atg4 was proposed as a protein fusion of Atg6 (and leading to its colocalization technique to detect autophagy . The assay is based with PI3P) was observed upon autophagy induction [101, on the cleavage by Atg4 of a luciferase protein fused to 102]. PI3P may be labelled in cells using a PI3P-binding Atg8/LC3 which, itself, is ﬁxed on actin cytoskeleton. In this peptide, FYVE fused to GFP . Quantiﬁcation of the system, actin-associated luciferase has a secretion signal and, accumulation of GFP-FYVE-labelled dots may also be used upon cleavage of Atg8/LC3 by Atg4, it is released from the as a tool to quantify autophagy activation upon starvation in cell. Luciferase activity can then be quantiﬁed in cellular mammalian cells (Yamaner Y. and Gozuacik D. unpublished supernatants reﬂecting Atg4 activity. Free luciferase can also data). Adaptations to the plant system may be possible be visualized in protein blots. Homologues of Atg4 are since orthologues of Atg6 and Vps34 are present in plants present in plants including Arabidopsis and rice; therefore, including Arabidopsis . this technique could be adapted to monitor Atg4 protease activity in plants. 4.2.3. Atg5 and Atg16. Atg5 as well as Atg16 was used as a selective marker to recognize autophagosome organization 4.2.6. Atg1 Activity. Atg1 is a serine/threonine kinase. Its centers (PAS). Since Atg5 dissociates after vesicle completion, activity correlated with autophagy induction [22, 27, 111– it will not label autophagosomes or lysosomes. The signal 113]. In S. cerevisiae, Atg1 autophosphorylation is dramat- could be detected as ﬂuorescent dots under microscope ically reduced upon starvation leading to autophagy . [38, 97]. A recent study used Atg16L as a new marker to In mammals, the function of Atg1 orthologues Ulk1 and detect autophagosome formation . Like Atg5, Atg16L Ulk2 seems to be controlled by autophosphorylation as well transiently associates with the surface of autophagosomes [113, 114]. Hence, Atg1 kinase activity and phosphorylation during their formation and forms punctate structures . status could be used as a new test of the autophagic activity Therefore, as Atg8/LC3, Atg5 and Atg16L, coupled with in cells, tissues, and extracts. In Arabidopsis thaliana genome, a ﬂuorophore or detected by immunoﬂuorescence using orthologues of the yeast genes coding for Atg1 kinase and speciﬁc antibodies, can be used to monitor autophagosome Atg13 have been identiﬁed [53, 115]. Therefore, measuring formation. As homologues of Atg5 and Atg16 exist in plants Atg1 activity could serve as a tool to monitor autophagy in (e.g., Arabidopsis, Z. mays) this technique might be useful in plants. plants studies as well. 4.2.4. Atg18. A mammalian orthologue of the yeast Atg18, 4.2.7. p62/SQSTM1. Sequestosome 1 (SQSTM1), also WIPI-1, was proposed as a marker for autophagy as well named ubiquitin-binding protein p62 (shortly p62), is a International Journal of Plant Genomics 7 stress-induced adaptor/marker protein that is a common autophagy-speciﬁc marker. These publications revealed that component of protein aggregates . p62 was shown MDC-positive structures colocalized only partially with to bind Atg8/LC3 proteins through its N-terminal region autophagosome markers in cells . Furthermore, in . p62/Atg8 interaction triggered degradation of protein autophagy-defective Atg5 knockout cells, MDC-positive dots aggregates by autophagy during which p62 itself was also were still observed . The ﬁgures labelled by MDC degraded [118, 119]. This observation led to the use of seem to be endosomes, lysosomes, and lamellar bodies p62 degradation as a molecular tool to detect autophagic . Therefore, MDC associates with acidic and lipid- activity [119–121]. As LC3 lipidation appears prior to p62 rich compartments and it does not discriminate between degradation, existence of a lag phase should be considered autophagosomes/autolysosomes and the aforementioned during the design of the experiments . Of note, it is still vesicular organelles. Hence, MDC staining has to be com- not known whether p62 is a general marker for autophagy bined with other techniques to avoid misinterpretations. and caution should be taken when using this technique with Whether MDC is also labelling nonautophagic structures in new autophagy-inducing stimuli. Our preliminary analyses plants needs careful investigation. revealed that there are no p62 orthologues in Arabidopsis. Yet, we cannot exclude the possibility that p62-like proteins 4.4. Biochemical Methods exist in plants. 4.4.1. Long-Lived Protein Degradation. Since autophagy is involved in the degradation of long-lived proteins, determi- 4.3. Tests of Lysosomal/Vacuolar Activity nation of their turnover appears to be an eﬃcient method 4.3.1. Lysotracker. Weakly basic amines selectively accumu- to monitor autophagy levels in cells. In the commonly used late in cellular compartments with low internal pH and technique, following metabolic labelling, degradation of all can be used to visualize acidic compartments such as long-lived proteins is measured. A radioactively labelled lysosomes/vacuoles. Lysotracker is a ﬂuorescent acidotropic amino acid such as valine or leucine can be used to probe used for labeling acidic organelles in live cells. It label newly synthesized proteins. Then cells are incubated consists of a ﬂuorophore linked to a weak base. Labelling with cold amino acids to allow short-lived proteins to be of acidic compartments by lysotracker is likely due to degraded. Finally, release of labelled amino acids resulting its protonation and retention in the membranes of these from the degradation of long-lived proteins is monitored organelles. Lytic compartment labelling methods such as . lysotracker staining must be used in combination with One major weakness of this technique is that autophagy more speciﬁc markers of autophagy in order to discrimi- is not the only mechanism of long-lived proteins degrada- nate autophagic activity from other events increasing lyso- tion. Autophagic and nonautophagic degradation of long- some/vacuole activity. Lysotracker staining method has been lived proteins should be distinguished by the use of used to monitor autophagy in various organisms including autophagy inhibitors such as 3-mehyladenine (3-MA) . Arabidopsis,tobacco,and barley [79, 80, 122]. An alternative nonradioactive method uses chromatography to monitor the amount of released unlabeled amino acids . 4.3.2. Acridine Orange (AO). AO is a ﬂuorescent basic dye Usage of metabolic labelling in plants was hindered by that has the ability to cross biological membranes. AO high compartmentalization of protein substrates and by the accumulates in acidic compartments, such as lysosomes fact that metabolite pools in plant cells are generally highly and vacuole, and becomes protonated and sequestered in dynamic . Recently developed techniques allowing their lumen. In acridine orange-stained cells, cytoplasm metabolic labeling of whole plants and plant cell cultures and nucleolus emit bright green ﬂuorescence, whereas may overcome these diﬃculties and allow quantiﬁcation of acidic compartments ﬂuoresce in bright red. Therefore, autophagy by long-lived protein degradation in plants [135– quantiﬁcation of the red ﬂuorescence reﬂects the degree of 137]. acidity and the volume of the cellular acidic compartments. Comparison of the ratio of green/red ﬂuorescence in cells, using ﬂuorescent microscopy or ﬂow cytometry, enables 4.4.2. Sequestration of Sugars. Radio-labelled sucrose or quantiﬁcation of the extent of autophagic degradation [123, raﬃnose, delivered to cytosol through electropermeabiliza- 124]. So far, to our knowledge, no study used AO as a plant tion, is sequestered in autophagic vesicles together with autophagy marker. engulfed cytosolic fragments. Accumulation of radioactivity in autophagic membrane fractions was used to measure 4.3.3. Monodansylcadaverine (MDC). The autoﬂuorescent autophagic activity [138, 139]. This method has its limita- substance monodansylcadaverine is commonly used to tions as well. For example, it cannot be used in yeast due to detect autophagy in plants and in other organisms [67, fast metabolism . Furthermore, injection of the labelled 125–127]. MDC is a weak base that is capable of crossing molecule can disturb cellular homeostasis, therefore, pre- biological membranes and concentrating in acidic com- cautions and extracontrols including determination of the partments . Although MDC was originally proposed metabolic equilibrium of the cell prior to the measurement to label autophagosomes and autolysosomes, recent studies are required. Sugar sequestration technique might be useful on mammalian autophagy brought out that it is not an in plant cell cultures studies and it needs to be tested. 8 International Journal of Plant Genomics Table 2: Advantages and disadvantages of techniques used to study autophagy. Technique Advantages Disadvantages Golden standard.Morphological characterization of Equipment and expertise required.Diﬃcult to Electron microscopy autophagosomes, autolysosomes and their cargo. make quantitative analyses. Rapid detection and quantiﬁcation of Dots do not always reﬂect autophagic Atg8/LC3 conjugation to autophagy.Amenable to high throughput techniques.Used activity.Molecular weight shift tests need lipid to create transgenic organisms for in vivo study of careful interpretation. autophagy. Other molecular markers Detection of various stages of autophagic vesicle (Atg5, Atg6, Atg16 and Most of them need further evaluation. formation. Atg18 detection) Reﬂects the activity of Vps34 kinase.Quantitative analysis PI3P accumulation in phenomena not directly PI3P detection possible. related to autophagy (vesicular transport). Atg1 and Atg4 activity Determination of enzymatic activity. So far no clear kinetic studies were published. Not all stimuli activate its P62/SQSTM1 degradation Activated especially by protein aggregates. degradation.Orthologue in plants? Determination and quantiﬁcation of autophagy-related Lysotracker and acridine Autophagosomes are not detected.Lytic activity lytic activity (lysosomal/vacuolar).FACscan analysis orange staining induced by other conditions as well. possible. Determination and quantiﬁcation of autophagy-related Not all autophagosomes are detected.Lytic MDC staining lytic activity (lysosomal/vacuolar). activity induced by other conditions as well. Nonspeciﬁc degradation of proteins by Long-lived protein Measures autophagic degradation of proteins.Kinetic mechanisms other than autophagy. Radioactive degradation measurements possible. technique. Measures autophagic sequestration phase.Quantiﬁcation Sequestration of sugars Sugars may be metabolized. may be possible. Phosphorylcholine Promising plant autophagy technique.Quantiﬁcation may Quantiﬁcation requires special equipment accumulation be possible. (NMR spectroscopy). Promising techniques for plant autophagy.Detection of Nonselective and selective Autophagy target proteins need further both sequestration and degradation phases.Quantiﬁcation degradation of proteins characterization. may be possible. Detection of autophagy target organelle Test of mitophagy or degradation.Various organelle-speciﬁc proteins or Quantiﬁcation not always possible. chloroplast autophagy organelle-tagged may be used. 4.4.3. Phosphorylcholine Accumulation. An assay to monitor between precursor and mature enzyme allows the detection autophagy in plants is based on the followup of phosphoryl- of autophagic activity in yeast cells. Nonselective degradation choline accumulation in cells. The technique was developed of marker proteins (especially those with an enzymatic in sycamore suspension cells cultures undergoing autophagy activity) might also be used in plants as autophagy detection upon sucrose starvation . Carbon starvation-activated methods. degradation of membrane lipids led to the accumulation of phosphorylcholine in the cytoplasm. Phosphorylcholine 4.5.2. Selective Autophagic Degradation of Proteins. Although accumulation correlated well with autophagy-induction and autophagy is generally considered as a nonselective phe- its quantiﬁcation by 31P-NMR spectroscopy was proposed as nomenon, some proteins appear to be selectively degraded a novel way of autophagy detection in plant cells. by autophagy. A GFP or DsRed construct, targeted to the chloroplast, and a GFP fusion of rubisco were transported 4.5. Other Techniques to the vacuole through autophagy [90, 142]. Rubisco is 4.5.1. Nonselective Degradation of Cytosolic Proteins. One of allocated most of the plant nitrogen and functions in carbon- the yeast techniques developed to monitor autophagy makes ﬁxation in chloroplasts. It is released from the chloroplasts use of an N-terminal truncated mutant of the yeast alkaline in structures called rubisco-containing bodies (RCBs) in phosphatase Pho8 . In contrast to the ER-localized order to provide nitrogen from the leaves to others organs. wild-type enzyme, the mutant form of pho8 lacking the RCB seem to overlap with autophagic vesicles, indicating N-terminal signal sequence (Pho8δ60), is delivered to the that rubisco is engulfed in autophagosomes and eventually vacuole by way of autophagy. Following entry to the vacuole, delivered to the vacuole. The process was dependent on ATG Pho8δ60 is cleaved at its C-terminus to produce the active genes underlining the autophagic character of the transport. alkaline phosphatase. 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