Progress 07/01/02 to 06/30/05
Outputs
Autophagy is an important route for the bulk removal of cytosolic proteins and organelles in all eukaryotes especially in cells/tissues undergoing apoptosis, or subjected to stress and starvation. Here, portions of the cytoplasm are engulfed in autophagic vesicles, which are then transported to the lysosome/vacuole for degradation by resident proteases. Although important during seed germination, growth during nutrient limiting conditions and senescence, the molecular mechanisms underpinning autophagy in plants are not well understood. Recently, a Tor kinase-activated protein modification system involving the ATG8 and ATG12 polypeptide tags has been shown to be essential for autophagy in yeast. Using the yeast genes as queries, we have identified an orthologous pathway in the plant Arabidopsis thaliana. Using a reverse genetic approach, we have generated a library of knockouts in many of the ATG loci involved in regulation of the conjugation pathways and nearly all of
the ATG loci involved in the two conjugation pathways. Phenotypic analyses of disruptions in ATG7 and ATG5, genes encoding the enzyme responsible for ATG8/12 activation and the target of ATG12 conjugation, respectively, indicate that the pathway is not essential for normal growth and development in Arabidopsis. However, the mutants are hypersensitive to N- and C-limiting media and undergo premature senescence, suggesting that the pathway has a role during nutrient-limiting conditions and extensive nutrient remobilization. For example, whereas wild-type can easily survive extended dark periods (10 days), the atp7 and atg5 mutants die quickly following reexposure to light (Thompson et al. 2005). Using these mutant lines and antibodies specific for several ATG proteins, we have begun examining the regulation and effects of starvation and senescence at both the protein and RNA levels. We have also developed fluorescently tagged versions of ATG8 and ATG5 to aid in tracking the formation of
autophagic vesicles. The formation of these vesicles and their subsequent delivery to the vacuole during starvation and senescence could easily be seen in intact plant cells using fluorescent confocal microscopy. We are now in the process of characterizing the a full set of mutants affecting the nine genes that encode ATG8, the two for ATG12, and the four genes that encode ATG1, the kinase that is activated by Tor and subsequently induces the conjugation of ATG8 and 12 to their respective targets.
Impacts
Autophagic protein degradation is an important nitrogen and carbon recycling process when plant are grown under nutrient limiting conditions or when plants attempt to lose cells/tissue via various programmed cell death events. Defining how plants achieve autophagy will aid in understanding how plant cope with nutrient limiting conditions and reuse carbon and nitrogen during senescence. The study of the ATG family of proteins offers for the first time a way to detect autophagic vesicle formation and their subsequent delivery into the vacuole for degradation. As a result, the dynamics of autophagy can now be visualized and examined under various stress and developmental states. Ultimately they may help identify methods to control protein turnover for the benefit of numerous agronomic processes, including seed germination, senescence, and survival under limiting growth conditions.
Publications
- Vierstra, R.D. 2003. The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends Plant Sci. 8: 135-142.
- Newbigin, E., and Vierstra, R.D. 2003. Plant reproduction ? sex and self denial. Nature 423: 229-230.
- Smalle, J., and Vierstra, R.D. 2004. The ubiquitin 26S proteasome proteolytic pathway. Ann. Rev. Plant Biology. 55: 555-590.
- Chae, Y.-K., H. Im, Q. Zhao, J.D. Doelling, R.D. Vierstra and J.L. Markley (2004) Prevention of aggregation after refolding by balanced stabilization-destabilization: production of the Arabidopsis thaliana protein (APG8a (At4g21980) for NMR structure determination. Protein Express. & Purif. 34: 280-2803.
- Thompson, A.R., and R.D. Vierstra (2005) Autophagic recycling: lessons from yeast help define the process in plants. Curr. Opin. Plant Biol. 8: 165-173.
- Downes, B.P., and R.D. Vierstra (2005) Post-translational regulation in plants employing a diverse set of polypeptide tags. Biochem. Soc. Trans. 32: 393-399.
- Thompson, A.R., J.H. Doelling, A, Suttangkakul, and R.D. Vierstra (2005) Autophagic nutrient recycling in Arabidopsis thaliana directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol. 138: 2097-2110.
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Progress 01/01/04 to 12/31/04
Outputs
Autophagy is an important route for the bulk removal of cytosolic proteins and organelles in all eukaryotes especially in cells/tissues undergoing apoptosis, or subjected to stress and starvation. Here, portions of the cytoplasm are engulfed in autophagic vesicles, which are then transported to the lysosome/vacuole for degradation by resident proteases. Although important during seed germination, growth during nutrient limiting conditions and senescence, the molecular mechanisms underpinning autophagy in plants are not well understood. Recently, a Tor kinase-activated protein modification system involving the ATG8 and ATG12 polypeptide tags has been shown to be essential for autophagy in yeast. Using the yeast genes as queries, we have identified an orthologous pathway in the plant Arabidopsis thaliana. In addition to genes encoding ATG8 and ATG12, proteins required for their activation, including the ATG1 kinase, and ultimate ligation to target proteins and/or
phospholipids have been identified. Nine distinct genes encoding APG8 are evident indicating that the Arabidopsis pathway may be more functionally diverse. Using a reverse genetic approach, we have generated a library of knockouts in many of the ATG loci involved in regulation of the conjugation pathways and nearly all of the ATG loci involved in the two conjugation pathways. Phenotypic analyses of disruptions in ATG7 and ATG5, genes encoding the enzyme responsible for ATG8/12 activation and the target of ATG12 conjugation, respectively, indicate that the pathway is not essential for normal growth and development in Arabidopsis. However, the mutants are hypersensitive to N- and C-limiting media and undergo premature senescence, suggesting that the pathway has a role during nutrient-limiting conditions and extensive nutrient remobilization. For example, whereas wild-type can easily survive extended dark periods (10 days), the atp7 and atg5 mutants die quickly following reexposure to
light. Using these mutant lines and antibodies specific for several ATG proteins, we have begun examining the regulation and effects of starvation and senescence at both the protein and RNA levels. We have also developed fluorescently tagged versions of ATG8 and ATG5 to aid in tracking the formation of autophagic vesicles. The formation of these vesicles. and their subsequent delivery to the vacuole during starvation and senescence could easily be seen in intact plant cells using fluorescent confocal microscopy. We are now in the process of purifying these vesicles to determine the types of proteins destined for degradation by autophagy.
Impacts
Autophagic protein degradation is an important nitrogen and carbon recycling process when plants are grown under nutrient limiting conditions or when plants attempt to lose cells/tissue via various programmed cell death events. Defining how plants achieve autophagy will aid in understanding how plants cope with nutrient limiting conditions and reuse carbon and nitrogen during senescence. The study of the ATG family of proteins offers, for the first time, a way to detect autophagic vesicle formation and their subsequent delivery into the vacuole for degradation. As a result, the dynamics of autophagy can now be visualized and examined under various stress and developmental states. Ultimately they may help identify methods to control protein turnover for the benefit of numerous agronomic processes, including seed germination, senescence, and survival under limiting growth conditions.
Publications
- Chae, Y.-K., H. Im, Q. Zhao, J.D. Doelling, R.D. Vierstra and J.L. Markley (2004) Prevention of aggregation after refolding by balanced stabilization-destabilization: production of the Arabidopsis thaliana protein (APG8a (At4g21980) for NMR structure determination. Protein Express. & Purif. 34: 280-2803.
- Thompson, A.R., and R.D. Vierstra (2005) Autophagic recycling: lessons from yeast help define the process in plants. Curr. Opin. Plant Biol. 8: 165-173.
- Downes, B.P., and R.D. Vierstra (2005) Post-translational regulation in plants employing a diverse set of polypeptide tags. Biochem. Soc. Trans. 32: 393-399.
- Thompson, A.R., J.H. Doelling, J.H., Suttangkakul, A., Walker, J.M. and Vierstra, R.D. 2005. Autophagic nutrient recycling in Arabidopsis thaliana directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol. (in press)
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Progress 01/01/03 to 12/31/03
Outputs
Autophagy is an important route for the bulk removal of cytosolic proteins and organelles in all eukaryotes especially in cells/tissues undergoing apoptosis, or subjected to stress and starvation. Here, portions of the cytoplasm are engulfed in autophagic vesicles, which are then transported to the lysosome/vacuole for degradation by resident proteases. Although important during seed germination, growth during nutrient limiting conditions and senescence, the molecular mechanisms underpinning autophagy in plants are not well understood. Recently, a Tor kinase-activated protein modification system involving the ATG8 and ATG12 polypeptide tags has been shown to be essential for autophagy in yeast. Using the yeast genes as queries, we have identified an orthologous pathway in the plant Arabidopsis thaliana. In addition to genes encoding APG8 and APG12, proteins required for their activation, including the APG1 kinase, and ultimate ligation to target proteins and/or
phospholipids have been identified. Nine distinct genes encoding APG8 are evident, indicating that the Arabidopsis pathway may be more functionally diverse. Using a reverse genetic approach, we have generated a library of knockouts in many of the APG loci involved in regulation of the conjugation pathways and nearly all of the ATG loci involved in the two conjugation pathways. Phenotypic analyses of disruptions in ATG7 and ATG5, genes encoding the enzyme responsible for ATG8/12 activation and the target of ATG12 conjugation, respectively, indicate that the pathway is not essential for normal growth and development in Arabidopsis. However, the mutants are hypersensitive to N- and C-limiting media and undergo premature senescence, suggesting that the pathway has a role during nutrient-limiting conditions and extensive nutrient remobilization. For example, whereas wild-type can easily survive extended dark periods (10 days), the atp7 and atg5 mutants die quickly following reexposure to
light. Using these mutant lines and antibodies specific for several ATG proteins, we have begun examining the regulation and effects of starvation and senescence at both the protein and RNA levels. We have also developed fluorescently tagged versions of ATG8 and ATG5 to aid in tracking the formation of autophagic vesicles. The formation of these vesicles and their subsequent delivery to the vacuole during starvation and senescence can easily be seen in intact plant cells using fluorescent confocal microscopy. Fluorescence imaging with additional molecular markers specific for various vesicles and intercellular compartments will also be used to track vesicle formation in the mutant background. These studies have and will continue to reveal the importance of autophagy in protein recycling and cellular maintenance in plants.
Impacts
Autophagic protein degradation is an important nitrogen and carbon recycling process when plants are grown under nutrient limiting conditions or when plants attempt to lose cells/tissue via various programmed cell death events. Defining how plants achieve autophagy will aid in understanding how plants cope with nutrient limiting conditions and reuse carbon and nitrogen during senescence. The study of the ATG family of proteins offers, for the first time, a way to detect autophagic vesicle formation and their subsequent delivery into the vacuole for degradation. As a result, the dynamics of autophagy can now be visualized and examined under various stress and developmental states. Ultimately they may help identify methods to control protein turnover for the benefit of numerous agronomic processes, including seed germination, senescence, and survival under limiting growth conditions.
Publications
- Vierstra, R.D. 2003. The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends Plant Sci. 8: 135-142.
- Newbigin, E., and Vierstra, R.D. 2003. Plant reproduction: sex and self denial. Nature 423: 229-230.
- Smalle, J., and Vierstra, R.D. 2004. The ubiquitin 26S proteasome proteolytic pathway. Ann. Rev. Plant Biology. 55: 555-590.
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Progress 07/01/02 to 12/31/02
Outputs
Autophagy is an important route for the bulk removal of cytosolic proteins and organelles in all eukaryotes especially in cells/tissues undergoing apoptosis, or subjected to stress and starvation. Here, portions of the cytoplasm are engulfed in autophagic vesicles, which are then transported to the lysosome/vacuole for degradation by resident proteases. Although important during seed germination, growth during nutrient limiting conditions and senescence, the molecular mechanisms underpinning autophagy in plants are not well understood. Recently, a Tor kinase-activated protein modification system involving the APG8 and APG12 polypeptide tags has been shown to be essential for autophagy in yeast. Using the yeast genes as queries, we have identified an orthologous pathway in the plant Arabidopsis thaliana. In addition to genes encoding APG8 and APG12, proteins required for their activation, including the APG1 kinase, and ultimate ligation to target proteins and/or
phospholipids have been identified. Nine distinct genes encoding APG8 are evident indicating that the Arabidopsis pathway may be more functionally diverse. Using a reverse genetic approach, we have generated a library of knockouts in many of the APG loci involved in regulation of the conjugation pathways and nearly all of the APG loci involved in the two conjugation pathways. Phenotypic analyses of disruptions in APG7 and APG5, genes encoding the enzyme responsible for APG8/12 activation and the target of APG12 conjugation, respectively, indicate that the pathway is not essential for normal growth and development in Arabidopsis. However, the mutants are hypersensitive to N- and C-limiting media and undergo premature senescence, suggesting that the pathway has a role during nutrient-limiting conditions and extensive nutrient remobilization. Using these mutant lines and antibodies specific for several APG proteins, we have begun examining the regulation and effects of starvation and
senescence at both the protein and RNA levels. We have also developed fluorescently tagged APG pathway proteins to aid in tracking the formation of autophagic vesicles and their subsequent delivery to the vacuole during starvation and senescence. Fluorescence imaging with additional molecular markers specific for various vesicles and intercellular compartments will also be used to track vesicle formation in the mutant background. These studies have and will continue to reveal the importance of autophagy in protein recycling and cellular maintenance in plants.
Impacts
The continued characterization of this important cellular process will allow us to further understand how plants deal with starvation and senescence. With the knowledge gained from this research we can potentially modify this system to help increase a plant's tolerance to harsh conditions and improve crop yield.
Publications
- Doelling JH, Walker JM, Friedman EM, Thompson AR, Vierstra RD. The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. J Biol Chem. 2002; 277(36):33105-14.
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