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Is the autophagy machinery an executioner of programmed cell death in plants?
Update
Trends in Plant Science
Vol.14 No.6
Letters
Is the autophagy machinery an executioner of programmed cell death in plants? Jean-Luc Cacas1 and Mark Diamond2 1 UMR 186 Re´sistance des Plantes aux Bioagresseurs (RPB), Equipe Me´canismes des Re´sistances, Institut de Recherche pour le De´veloppement, 911 Avenue Agropolis, B.P. 64501, 34394 Montpellier Cedex 5, France 2 Biotech Center, Rutgers The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ 08901, USA
In a recent issue of Trends in Plant Science, Andrew J. Love et al. [1] provided an interesting point of view regarding regulatory overlap between apoptotic-like and autophagic programmed cell death (PCD). Taking into account that the two forms of cell death share cytological and biochemical features, the authors reasonably hypothesized that they might also possess a common set of molecular regulators. The authors further postulated that the nature of the stimuli would determine the cell death rate and, consequently, whether apoptotic-like (rapid) or autophagic (slow) PCD would be engaged. Although we do acknowledge that the overall analysis is valuable and original, we became concerned when autophagy was described as the process responsible for cell demise during ‘autophagic PCD’ (see Box 1 and Glossary in Ref. [1]). Historically, autophagic cell death, also known as type II cell death, has been defined using morphological criteria: it was characterized by the lack of chromatin condensation and a massive autophagic vacuolization of the cytoplasm [2]. Therefore, this definition does not mention any causative role for autophagy in the cell death process. Furthermore, even though the term ‘autophagic cell death’ might intuitively imply such an idea, to our knowledge this has not yet been demonstrated in plants. The question as to whether the cytoplasmic vesiculation taking place during ‘autophagic cell death’ actually serves to kill cells is controversial and has long been discussed in the animal biology field. It remained unanswered until recently, when apoptosis-deficient mammalian cell lines were engineered to be impaired in the formation of autophagic structures, so-called autophagosomes. Silencing AUTOPHAGY (ATG) genes indeed revealed a concomitant decrease in both the cell death rate and number of autophagosome positive cells upon various stimuli [3,4], indicating that autophagy could be employed in these specific conditions as a tool dedicated to cellular dismantling. Besides these in vitro studies, there are only two examples published thus far showing that the successful execution of PCD requires functional autophagy machinery in vivo: work on degradation of salivary gland cells in Drosophila [5] and reports on cell death associated with spore germination of the rice blast fungus [6]. In plants, as mentioned by Love et al. (see Figure 1 in Ref. [1]), it is true that several developmental and stressinduced PCD models seem to display an autophagic stereotype. However, the autophagic nature of the vacuolization Corresponding author: Cacas, J.-L. ([email protected]).
accompanying cell death is not always investigated or properly demonstrated in the literature. Autophagic structures, unlike other vesicles, possess a double membrane. Thus, a bona fide method used by researchers to distinguish among vesicles relies on the use of electron microscopy [2]. Tracking systems using green fluorescent protein or dyes have also been proven to be instrumental in visualizing autophagosomes in planta [7]. Utilizing these technologies would surely be useful for ascertaining PCD type, although it is not sufficient to clearly establish when autophagy contributes to cell death as an executioner component. Genetic approaches will definitively bring the necessary proof. Since the identification of ATG sequence homologues in plant genomes, such analyses have been undertaken. However, to date, there is only evidence for ATG-dependent autophagy as a pro-survival mechanism. In ATGsilenced tobacco lines (ATG3, ATG7 or ATG6), bacterial and viral infections triggered an uncontrolled hypersensitive cell death that spread systemically, ultimately killing the whole plant [8]. Likewise, Arabidopsis thaliana knockout and knockdown lines (ATG7, ATG9, ATG18a) exhibited an accelerated senescence phenotype comprising earlier cell death [9–11]. Given that autophagy can have a dual role in animals, pro- or anti-PCD, these results are not surprising, and it is tempting to speculate that conditions in which ATG-dependent autophagy causes cell death still have to be found in plants. Unravelling additional autophagy mediators might thus be of crucial importance for understanding the relationship between autophagy and PCD and advancing the debate on ‘autophagic cell death’. Acknowledgements We wish to warmly thank Michel Nicole for critical reading and helpful discussions.
References 1 Love, A.J. et al. (2008) Timing is everything: regulatory overlap in plant cell death. Trends Plant Sci. 13, 589–595 2 Kroemer, G. et al. (2005) Classification of cell death: recommendations of the Nomenclature Committee on Cell Death. Cell Death Differ. 12, 1463–1467 3 Yu, L. et al. (2004) Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500–1502 4 Shimizu, S. et al. (2004) Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nat. Cell Biol. 6, 1221–1228 5 Berry, D.L. and Baehrecke, E.H. (2007) Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell 131, 1137–1148 299
Update 6 Veneault-Fourrey, C. et al. (2006) Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 312, 580–583 7 Bassham, D.C. (2007) Plant autopahgy – more than a starvation response. Curr. Opin. Plant Biol. 10, 587–593 8 Liu, Y. et al. (2005) Autophagy regulates programmed cell death during the plant innate immune response. Cell 121, 567–577 9 Doelling, J.H. et al. (2002) The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. J. Biol. Chem. 277, 33105–33114
Trends in Plant Science Vol.14 No.6 10 Hanaoka, H. et al. (2002) Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol. 129, 1181–1193 11 Xiong, Y. et al. (2005) AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J. 42, 535–546 1360-1385/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2009.02.008 Available online 4 May 2009
Letters Response
Response to Cacas and Diamond: Is the autophagy machinery an executioner of programmed cell death in plants? Andrew J. Love, Joel J. Milner and Ari Sadanandom Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK
We are grateful for the interest that Jean-Luc Cacas and Mark Diamond have shown towards our Trends in Plant Science opinion article on the timing and co-ordinate regulation of plant cell death [1]. They identified a potential catachresis in our definition of autophagic programmed cell death (PCD), whereby they suggest that the autophagy observed in fatally committed cells might not directly induce cell death. Our initial definition of ‘autophagic cell death’ was more intended to highlight that autophagy was a predominant feature of this type of cell death, irrespective of its role in the death process. Although our opinion article was written from a broad signalling perspective, we are glad that it generated a response from those interested in autophagy. Autophagy is a tightly controlled process, and investigation of autophagy frequently produces conflicting results. For example, knocking out genes regulating autophagy might either promote or inhibit autophagic pathways; which in turn might inhibit or promote the cell death processes. This probably reflects the complexities of potential feedback regulation and the type of studies carried out. Here, we briefly reiterate the evidence for pro-life and pro-death roles of autophagy. Cacas and Diamond [2] quite correctly ingeminate that autophagy has a pro-life function. This has been well characterized in a plethora of different cell types, such as human haemopoietic cells [3], and organisms, such as Caenorhabditis elegans [4], Dictyostelium discoideum [5] and tobacco (Nicotiana tabacum) [6]. In these cases it was demonstrated that inhibition of autophagy by ablation or silencing of AUTOPHAGY (ATG) genes led to cell death during hormone and nutrient deprivation and pathogen attack. The pro-life function of autophagy is possibly due to its capacity to improve cellular fitness via a controlled vacuolar recycling and redistribution of nutrients. This process is known to inhibit apoptosis-like cell death in plants. Corresponding author: Sadanandom, A. ([email protected]).
300
Conversely, there is also considerable evidence to suggest that autophagy might have a ‘pro-death’ function. Cacas and Diamond [2] stated that ‘there are only two examples published thus far showing that the successful execution of PCD requires functional autophagy machinery in vivo: the work on degradation of salivary gland cells in Drosophila [7] and reports on cell death associated with spore germination of the rice blast fungus [8]’. In fact, there have been many studies carried out on C. elegans [9], HeLa cells [10], mouse embryonic fibroblasts [11] and murine fibrosarcoma cells [12] in which the genetic or chemical ablation of autophagy components suppresses cell death, thereby illustrating that in many situations autophagy is a requirement of PCD. Because autophagy has a pro-death role in many different organisms, it is compelling to assume that this also applies to plants, and indeed there is some evidence to support this. Knocking out atg7 and atg9 in Arabidopsis leads to either normal or enhanced levels of autophagy, with the concomitant development of premature senescence (see Ref. [2]). However, the mechanics behind this process are poorly understood at present. It is possible that the outcome of autophagy, whether it be pro-life or pro-death, is dependent on the method of influencing ATG gene regulation (i.e. genetic knockouts or gene silencing) and the particular treatment applied to the organism. In addition, it might be an oversimplification to assume that the pro-life or pro-death phenotypes that result from the knockout of ATG genes are caused by suppression of the autophagic process because ATG genes might have diverse roles in cellular regulation. For autophagy to be properly correlated to pro-life or -death functions, it has to be measured in an appropriate manner, which is often difficult because there are many different assays, each with their own caveats. Recently, a large consortium of experts published guidelines for accurately measuring autophagy, which should enable a more thorough interpretation of experimental results [13]. This, coupled with the identification of
Trends in Plant Science
Vol.14 No.6
Letters
Is the autophagy machinery an executioner of programmed cell death in plants? Jean-Luc Cacas1 and Mark Diamond2 1 UMR 186 Re´sistance des Plantes aux Bioagresseurs (RPB), Equipe Me´canismes des Re´sistances, Institut de Recherche pour le De´veloppement, 911 Avenue Agropolis, B.P. 64501, 34394 Montpellier Cedex 5, France 2 Biotech Center, Rutgers The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ 08901, USA
In a recent issue of Trends in Plant Science, Andrew J. Love et al. [1] provided an interesting point of view regarding regulatory overlap between apoptotic-like and autophagic programmed cell death (PCD). Taking into account that the two forms of cell death share cytological and biochemical features, the authors reasonably hypothesized that they might also possess a common set of molecular regulators. The authors further postulated that the nature of the stimuli would determine the cell death rate and, consequently, whether apoptotic-like (rapid) or autophagic (slow) PCD would be engaged. Although we do acknowledge that the overall analysis is valuable and original, we became concerned when autophagy was described as the process responsible for cell demise during ‘autophagic PCD’ (see Box 1 and Glossary in Ref. [1]). Historically, autophagic cell death, also known as type II cell death, has been defined using morphological criteria: it was characterized by the lack of chromatin condensation and a massive autophagic vacuolization of the cytoplasm [2]. Therefore, this definition does not mention any causative role for autophagy in the cell death process. Furthermore, even though the term ‘autophagic cell death’ might intuitively imply such an idea, to our knowledge this has not yet been demonstrated in plants. The question as to whether the cytoplasmic vesiculation taking place during ‘autophagic cell death’ actually serves to kill cells is controversial and has long been discussed in the animal biology field. It remained unanswered until recently, when apoptosis-deficient mammalian cell lines were engineered to be impaired in the formation of autophagic structures, so-called autophagosomes. Silencing AUTOPHAGY (ATG) genes indeed revealed a concomitant decrease in both the cell death rate and number of autophagosome positive cells upon various stimuli [3,4], indicating that autophagy could be employed in these specific conditions as a tool dedicated to cellular dismantling. Besides these in vitro studies, there are only two examples published thus far showing that the successful execution of PCD requires functional autophagy machinery in vivo: work on degradation of salivary gland cells in Drosophila [5] and reports on cell death associated with spore germination of the rice blast fungus [6]. In plants, as mentioned by Love et al. (see Figure 1 in Ref. [1]), it is true that several developmental and stressinduced PCD models seem to display an autophagic stereotype. However, the autophagic nature of the vacuolization Corresponding author: Cacas, J.-L. ([email protected]).
accompanying cell death is not always investigated or properly demonstrated in the literature. Autophagic structures, unlike other vesicles, possess a double membrane. Thus, a bona fide method used by researchers to distinguish among vesicles relies on the use of electron microscopy [2]. Tracking systems using green fluorescent protein or dyes have also been proven to be instrumental in visualizing autophagosomes in planta [7]. Utilizing these technologies would surely be useful for ascertaining PCD type, although it is not sufficient to clearly establish when autophagy contributes to cell death as an executioner component. Genetic approaches will definitively bring the necessary proof. Since the identification of ATG sequence homologues in plant genomes, such analyses have been undertaken. However, to date, there is only evidence for ATG-dependent autophagy as a pro-survival mechanism. In ATGsilenced tobacco lines (ATG3, ATG7 or ATG6), bacterial and viral infections triggered an uncontrolled hypersensitive cell death that spread systemically, ultimately killing the whole plant [8]. Likewise, Arabidopsis thaliana knockout and knockdown lines (ATG7, ATG9, ATG18a) exhibited an accelerated senescence phenotype comprising earlier cell death [9–11]. Given that autophagy can have a dual role in animals, pro- or anti-PCD, these results are not surprising, and it is tempting to speculate that conditions in which ATG-dependent autophagy causes cell death still have to be found in plants. Unravelling additional autophagy mediators might thus be of crucial importance for understanding the relationship between autophagy and PCD and advancing the debate on ‘autophagic cell death’. Acknowledgements We wish to warmly thank Michel Nicole for critical reading and helpful discussions.
References 1 Love, A.J. et al. (2008) Timing is everything: regulatory overlap in plant cell death. Trends Plant Sci. 13, 589–595 2 Kroemer, G. et al. (2005) Classification of cell death: recommendations of the Nomenclature Committee on Cell Death. Cell Death Differ. 12, 1463–1467 3 Yu, L. et al. (2004) Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500–1502 4 Shimizu, S. et al. (2004) Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nat. Cell Biol. 6, 1221–1228 5 Berry, D.L. and Baehrecke, E.H. (2007) Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell 131, 1137–1148 299
Update 6 Veneault-Fourrey, C. et al. (2006) Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 312, 580–583 7 Bassham, D.C. (2007) Plant autopahgy – more than a starvation response. Curr. Opin. Plant Biol. 10, 587–593 8 Liu, Y. et al. (2005) Autophagy regulates programmed cell death during the plant innate immune response. Cell 121, 567–577 9 Doelling, J.H. et al. (2002) The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. J. Biol. Chem. 277, 33105–33114
Trends in Plant Science Vol.14 No.6 10 Hanaoka, H. et al. (2002) Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol. 129, 1181–1193 11 Xiong, Y. et al. (2005) AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J. 42, 535–546 1360-1385/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2009.02.008 Available online 4 May 2009
Letters Response
Response to Cacas and Diamond: Is the autophagy machinery an executioner of programmed cell death in plants? Andrew J. Love, Joel J. Milner and Ari Sadanandom Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK
We are grateful for the interest that Jean-Luc Cacas and Mark Diamond have shown towards our Trends in Plant Science opinion article on the timing and co-ordinate regulation of plant cell death [1]. They identified a potential catachresis in our definition of autophagic programmed cell death (PCD), whereby they suggest that the autophagy observed in fatally committed cells might not directly induce cell death. Our initial definition of ‘autophagic cell death’ was more intended to highlight that autophagy was a predominant feature of this type of cell death, irrespective of its role in the death process. Although our opinion article was written from a broad signalling perspective, we are glad that it generated a response from those interested in autophagy. Autophagy is a tightly controlled process, and investigation of autophagy frequently produces conflicting results. For example, knocking out genes regulating autophagy might either promote or inhibit autophagic pathways; which in turn might inhibit or promote the cell death processes. This probably reflects the complexities of potential feedback regulation and the type of studies carried out. Here, we briefly reiterate the evidence for pro-life and pro-death roles of autophagy. Cacas and Diamond [2] quite correctly ingeminate that autophagy has a pro-life function. This has been well characterized in a plethora of different cell types, such as human haemopoietic cells [3], and organisms, such as Caenorhabditis elegans [4], Dictyostelium discoideum [5] and tobacco (Nicotiana tabacum) [6]. In these cases it was demonstrated that inhibition of autophagy by ablation or silencing of AUTOPHAGY (ATG) genes led to cell death during hormone and nutrient deprivation and pathogen attack. The pro-life function of autophagy is possibly due to its capacity to improve cellular fitness via a controlled vacuolar recycling and redistribution of nutrients. This process is known to inhibit apoptosis-like cell death in plants. Corresponding author: Sadanandom, A. ([email protected]).
300
Conversely, there is also considerable evidence to suggest that autophagy might have a ‘pro-death’ function. Cacas and Diamond [2] stated that ‘there are only two examples published thus far showing that the successful execution of PCD requires functional autophagy machinery in vivo: the work on degradation of salivary gland cells in Drosophila [7] and reports on cell death associated with spore germination of the rice blast fungus [8]’. In fact, there have been many studies carried out on C. elegans [9], HeLa cells [10], mouse embryonic fibroblasts [11] and murine fibrosarcoma cells [12] in which the genetic or chemical ablation of autophagy components suppresses cell death, thereby illustrating that in many situations autophagy is a requirement of PCD. Because autophagy has a pro-death role in many different organisms, it is compelling to assume that this also applies to plants, and indeed there is some evidence to support this. Knocking out atg7 and atg9 in Arabidopsis leads to either normal or enhanced levels of autophagy, with the concomitant development of premature senescence (see Ref. [2]). However, the mechanics behind this process are poorly understood at present. It is possible that the outcome of autophagy, whether it be pro-life or pro-death, is dependent on the method of influencing ATG gene regulation (i.e. genetic knockouts or gene silencing) and the particular treatment applied to the organism. In addition, it might be an oversimplification to assume that the pro-life or pro-death phenotypes that result from the knockout of ATG genes are caused by suppression of the autophagic process because ATG genes might have diverse roles in cellular regulation. For autophagy to be properly correlated to pro-life or -death functions, it has to be measured in an appropriate manner, which is often difficult because there are many different assays, each with their own caveats. Recently, a large consortium of experts published guidelines for accurately measuring autophagy, which should enable a more thorough interpretation of experimental results [13]. This, coupled with the identification of