Abstract
Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.
Highlights
ATP is a ubiquitous, energy-rich molecule of fundamental importance in living organisms
Establishing the Experimental System—To identify eATPregulated proteins with a putative function in cell death regulation, we used Arabidopsis cell cultures treated with fumonisin B1 (FB1) or FB1ϩATP
Exogenous ATP treatment can be used as a filter to identify the subset of FB1-induced genes/proteins whose expression and abundance is altered in response to the specific depletion of extracellular ATP (eATP)
Summary
Plant Material and Growth Conditions—Cell suspension cultures of Arabidopsis thaliana var. Protein Labeling and Gel Electrophoresis—Protein samples from four biological replicates of each treatment were labeled with CyDyes (GE Healthcare, Buckinghamshire, UK) as described before [35]. Manually inspected spots present in all replicate gels and displaying a significant (p Յ 0.05) FB1-induced change in abundance of a minimum of 20% (TSP) or 50% (microsomal protein) were selected for further analyses. The response of these spots to ATP added after FB1 was analyzed by statistical comparison of FB1ϩATP average with FB1 only average. Symptom development was visually monitored and photographs taken 4 days following infiltration
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