Characterization of 14-3-3 [chi] phosphorylation in phosphorus stress in Arabidopsis thaliana and investigation of acetyl-coA carboxylase regulation using quantitative mass spectrometry
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Oilseed development involves coordination and regulation of a complex metabolic network for efficient production of storage compounds, including fatty acids, proteins, and carbohydrates. Recent advances in mass spectrometry have enabled identification of thousands of proteins and post-translational modifications in a variety of oilseed species, however, much remains to be known about their regulatory roles in seed development. A current focus in oilseed research is regulatory elucidation of these proteins and modifications, which can ultimately be applied to genetic engineering applications for higher production of storage compounds. This work involves characterization of an Arabidopsis 14-3-3 isoform [chi], an abundant and phosphorylated protein identified in seed development. 14-3-3 [chi] involvement in phosphorus stress was demonstrated and used to elucidate the function of phosphorylation in vivo. Results indicate that phosphorylation of 14-3-3 [chi] affects dimerization and interactions with client proteins. In addition, a targeted mass spectrometry method was employed to absolutely quantify heteromeric acetyl-CoA carboxylase (ACCase) subunits and putative inhibitory BADC proteins during feedback inhibition and temporal regulation. Quantitation revealed ACCase expression is negatively regulated during feedback inhibition by Tween feeding, while BADC expression displayed a positive response. These results suggest BADC proteins could inhibit the ACCase complex during feedback inhibition. ACCase and BADC expression was also found to be temporally regulated during Arabidopsis silique development. Furthermore, abundance of an ACCase subunit, [alpha]-CT, was low in Arabidopsis siliques, suggesting that it could be a limiting component of the complex.
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