Progress 08/01/02 to 07/31/06
Outputs
This project has focused on the role of vacuolar autophagy in starvation and other environmental stress responses in Arabidopsis thaliana. Initially, global gene expression changes were analyzed upon autophagy induction during sucrose starvation in Arabidopsis suspension cultured cells using the Affymetrix 22,800 gene chip. These data allowed us to propose a model for the integration of starvation responses, including autophagy and other stress responses, metabolic changes and eventual cell death. One difficulty in studying autophagy in whole plants has been the lack of a simple and convenient assay for the process. We therefore have developed such an assay that can detect autophagy in intact, live plants, using a fluorescent lipophilic dye that accumulates in autophagosomes. We have shown that this dye label also co-localizes in starved plant cells with a protein (AtATG8e) identified from our transcriptional analysis above (as a green fluorescent protein fusion) that
is a predicted autophagosome marker. Using these two markers, we have analyzed the conditions under which autophagy is induced in plants, and shown that autophagosomes form not only during starvation (both carbon and nitrogen) but also during leaf senescence. A second gene upregulated during starvation is AtATG18a and transgenic lines expressing a reduced level of AtATG18a mRNA are hypersensitive to carbon and nitrogen deficiency and display early senescence. These phenotypes are consistent with our determination of the conditions in which autophagy is induced in plants. Using our assay described above, we have shown that autophagosomes do not form in this mutant, implicating AtATG18a in autophagosome formation. We are now expanding our study of autophagy to identify other stress conditions in which this process is important for plant growth and survival. We have discovered that autophagy is also induced by oxidative stress, and that plants defective in autophagy are more sensitive to
oxidative stress than wild type plants. One underlying cause for this sensitivity is that autophagy delivers oxidized proteins to the vacuole for degradation.
Impacts
This research has provided insight into the mechanism and regulation of autophagy and genes required for autophagy have been identified. As autophagy is required for stress responses and for appropriate timing of senescence, modification of the autophagy pathway is expected to lead to improvements in stress tolerance in the field, impacting crop yield. The role of autophagy in nutrient remobilization may also lead to environmental impacts due to possible reductions in fertilizer application.
Publications
- Bassham DC, Laporte M, Marty F, Moriyasu Y, Ohsumi Y, Olsen LJ and Yoshimoto K. 2006. Autophagy in development and stress responses of plants. Autophagy 2: 2-11.
- Xiong Y, Contento AL and Bassham DC. 2005. AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J 42: 535-546.
- Contento AL, Xiong Y and Bassham DC. 2005. Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-ATG8e fusion protein. Plant J 42: 598-608.
- Contento AL, Kim SJ and Bassham DC. 2004. Transcriptome profiling of the response of Arabidopsis thaliana suspension culture cells to sucrose starvation. Plant Physiol 135: 2330-2347.
- Bassham DC. 2002. Golgi-independent trafficking of macromolecules to the plant vacuole. Adv Bot Res 38: 65-92.
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Progress 10/01/04 to 09/30/05
Outputs
Two markers have been developed that can be used to monitor autophagy in Arabidopsis, in suspension cells, protoplasts and whole seedlings. The first is monodansylcadaverine (MDC), a fluorescent dye that specifically stains autophagosomes. The second is a GFP-AtATG8e fusion protein that localizes to autophagosomal membranes upon induction of autophagy. AtATG8e was identified as a homolog of the autophagy-specific yeast protein Atg8 that was induced upon sucrose starvation of Arabidopsis suspension cells, as determined by Affymetrix GeneChip transcriptional profiling. These two markers were used to demonstrate that autophagy is induced during sucrose starvation of suspension cells and seedlings, nitrogen deficiency in seedlings, and during early stages of leaf senescence. Another gene identified as upregulated during starvation shows sequence similarity to the yeast autophagy gene Atg18 and is designated AtATG18a. AtATG18a is a member of a small gene family in
Arabidopsis, but is the only member of the family to be upregulated upon both starvation and senescence, conditions which induce autophagy. RNA interference was used to decrease AtATG18a expression in transgenic Arabidopsis plants, without affecting expression of other members of the family. AtATG18a RNAi lines were shown to be hypersensitive to sucrose and nitrogen starvation and showed premature senescence, suggestive of a role in autophagy. This was confirmed by MDC staining, demonstrating that the RNAi lines are unable to produce autophagosomes either during starvation or senescence.
Impacts
The autophagy markers developed are expected to be useful to many researchers in their ability to monitor autophagy in Arabidopsis in vivo in a non-destructive manner. They are also likely to be useful in other plant species, including major crop species. The development of the AtATG18a RNAi lines now provides a tool for analysis of the physiological consequences of altering the autophagy pathway in plants.
Publications
- Xiong Y, AL Contento and DC Bassham. 2005. AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J 42: 535-546.
- Contento AL, Y Xiong and DC Bassham. 2005. Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-ATG8e fusion protein. Plant J 42: 598-608.
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Progress 10/01/03 to 09/30/04
Outputs
The overall aims of this project are to determine the physiological role of autophagy in the response of Arabidopsis plants to nutrient starvation and to identify genes involved in this process. The approach taken is to use Affymetrix GeneChips to identify genes whose mRNA levels increase in Arabidopsis suspension cultures during sucrose starvation. A subset of the genes identified are being characterized further to determine their function in the starvation response. We expect that this research will provide information for use in the modification of crop plants to enhance their resistance to environmental stress conditions. Autophagy is induced in Arabidopsis cells after 24-48 hours of sucrose starvation (Bassham, 2002). RNA was isolated from Arabidopsis suspension cells after 0, 24 or 48 hours of sucrose starvation and transcriptional profiling was performed using the ATH1 GeneChip array containing 22,810 probe sets. A significant increase in transcript levels was
observed for 343 genes within 48 hours of starvation, including genes predicted to function in the recycling of cellular components and nutrient scavenging for maintaining cell function, the protection of the cell from death through activation of various defense and stress response pathways, and regulation of these processes by specific protein kinases and transcription factors (Contento et al., 2004). Our data support a model in which plant cells initiate a coordinated response of nutrient mobilization at the onset of sucrose depletion that is able to maintain cell viability for up to 48 hours. After this point, genes potentially involved in cell death increase in expression whereas those functioning in translation and replication decrease, leading to a decrease in culture viability and activation of cell death programs. Based on these results, we have chosen several genes for further study of their role in nutrient stress responses. One example is a homolog of yeast Atg8, a
lipid-modified protein that is required for autophagy and is a well-characterized marker for autophagosomes. The Arabidopsis genome encodes 9 potential Atg8 homologs, but only one (AtATG8e) was upregulated under our starvation conditions. We have generated a GFP-AtATG8e fusion and shown that it labels autophagosomes in Arabidopsis protoplasts, and can therefore be used as an autophagy marker in plant cells.
Impacts
The research conducted thus far in this project has given us a detailed picture of gene expression patterns during sucrose starvation in Arabidopsis. Sucrose deficit is likely to occur in plants in the field under a wide variety of conditions that limit photosynthetic efficiency; therefore, the genes identified as responding to starvation may be critical in plant survival under stress conditions. The expression data both predicts the cellular pathways and processes that are important for nutrient stress responses, and provides a rich source of targets for genetic modification of these pathways. Modification of autophagy and other processes has the potential to lead to changes in the yield of crop plants by improving growth under the sub-optimal conditions encountered in the field. In addition to potential impacts on agriculture, this research has also led to the identification of markers for autophagy in whole plants. This provides an invaluable tool for the further
study of this process, and will allow us to rapidly evaluate the effect of mutations in potential autophagy genes.
Publications
- Bassham DC (2002) Golgi-independent trafficking of macromolecules to the plant vacuole. Adv. Bot. Res. 38: 65-92.
- Contento, AL, Kim, S-K and Bassham, DC (2004) Transcriptome profiling of the response of Arabidopsis suspension culture cells to sucrose starvation. Plant Physiol. 135: 2330-2347.
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Progress 08/01/02 to 07/31/03
Outputs
DETERMINATION OF THE TIME COURSE OF CHANGES IN GENE EXPRESSION IN ARABIDOPSIS SUSPENSION CULTURES IN RESPONSE TO SUCROSE STARVATION. We have used gene expression analysis to isolate genes required for plant nutrient stress responses, and thus to define potential targets for modification of these responses to improve growth under nutrient stress condiitons. To identify genes that show an increase in expression during starvation, RNA was isolated from Arabidopsis suspension cells after 0, 24 or 48 hours of sucrose starvation and used to probe Affymetrix GeneChips to analyze changes in mRNA abundance due to the nutrient stress. Approximately 500 genes were identified whose RNA increases in abundance during starvation. Those genes with a known or predicted function were classified according to the MIPS functional classification. A large number of the genes identified are predicted to be involved in metabolism, in particular in catabolic pathways such as amino acid
degradation. We hypothesize that these genes function in pathways for the use of alternative energy and carbon sources to enable cell survival when sugar is limiting. A number of genes encoding membrane transporters, for sugars and other nutrients, are also upregulated during starvation, presumably to increase the efficiency of scavenging nutrients from the environment. Another large group of genes were identified as being involved in cell rescue and defense. These include genes that were already known to be induced during various abiotic and biotic stresses, and may represent general stress response pathways. Several genes previously described as being involved in pathogen responses are evident, as are genes encoding reactive oxygen detoxification enzymes. These genes are likely to function in the protection of the plant cells under stress. Many genes encoding potential transcription factors and signal transduction components increased in expression during starvation. These genes are
of particular interest, as they may encode proteins that function in the regulation of autophagy and other nutrient stress responses. Potential transcription factors include representatives of the WRKY, myb and bZIP families; interestingly, only a few members of these large families show an increase in our experiments. Other components include several protein kinases that could play a role in stress signaling pathways. Finally, several genes that may function in membrane dynamics and structure have been identified, including activating proteins for small GTPases, a phosphatidylinositol kinase and a vacuolar sorting receptor. The encoded proteins may function in the dramatic changes in cell structure and membrane organization that occur during autophagy.
Impacts
The research conducted thus far in this project has given us a detailed picture of gene expression patterns during sucrose starvation in Arabidopsis. Sucrose deficit is likely to occur in plants in the field under a wide variety of conditions that limit photosynthetic efficiency; therefore, the genes identified as responding to starvation may be critical in plant survival under stress conditions. The expression data both predicts the cellular pathways and processes that are important for nutrient stress responses, and provides a rich source of targets for genetic modification of these pathways. Modification of autophagy and other processes has the potential to lead to changes in the yield of crop plants by improving growth under the sub-optimal conditions encountered in the field.
Publications
- Bassham DC. 2002. Golgi-independent trafficking of macromolecules to the plant vacuole. Advances In Botanical Research 38: 65-92.
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