Characterization of the 14-3-3 client protein interactome from developing arabidopsis seed and regulation by protein phosphorylation
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Understanding the regulatory mechanisms controlling reserve deposition during seed development is an academic endeavor with the potential for socioeconomic impact. Diversity in seed storage reserves among plant species as well as species ecotypes and hybrids suggests molecular and biochemical processes underlie the natural variation in seed composition. An understanding of these mechanisms could directly influence our ability to control oil, protein, and carbohydrate levels within seeds. While Arabidopsis is not a commercial crop, it has become a model organism for studying oilseed development. This is due in large part to its high oil content, genetic resources, and relationship to the agronomic crop, Brassica napus (a.k.a., rapeseed, canola). The application of high-throughput transcriptomic and proteomic approaches to seed development has cataloged the expression of thousands of genes, providing molecular insight into seed development. One class of regulatory proteins found to be highly expressed throughout seed filling in soybean, rapeseed, castor, and Arabidopsis was the 14-3-3 protein family. These proteins accounted for as much as 2% of the total proteome during seed development based upon two-dimensional (2D) gel analyses. Furthermore, a 2D difference gel electrophoresis (DIGE) based proteomic investigation of a near-isogenic sunflower line bred for high oleic acid content showed increased 14-3-3 expression levels when compared to its parental line. Due to their prominence and status of global 'phosphoregulators', we examined the role of 14-3-3 proteins in developing Arabidopsis seed by elucidating their protein associations. Here, we describe the 14-3-3 developing seed interactome of two phylogenetically unique paralogs, chi and epsilon. Interestingly, both displayed unique client protein (binding partners) preferences, while primarily interacting with enzymes of primary metabolism, including glycolysis and de novo fatty acid synthesis. Together, these studies suggest 14-3-3 proteins play a regulatory role in seed filling and maturation by influencing carbon assimilatory networks. A phosphoproteomic screen of developing rapeseed provided further evidence of 14-3-3 proteins involvement in seed development. The identification of 14-3-3 chi and epsilon phosphorylation sites suggest this post-translational modification may have a functional role in seed development. In plants, 14-3-3 phosphorylation has been identified on multiple sites in various tissues. However, an understanding of the kinases capable of phosphorylating 14-3-3 proteins is limited. Phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases (CPKs) was monitored because 14-3-3 proteins modulate CPK activity and the activity of CPK substrates. We therefore applied a quantitative mass spectrometry (MS)-based assay to monitor 14-3-3 phosphorylation by multiple clades of CPKs. In total, seven 14-3-3 phosphorylation sites were identified, one of which was previously identified in vivo. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analyzed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF hand domain of CPK28, suggesting CPKs may function as dual-specificity kinases. Of all CPKs assayed, CPK28, which was autophosphorylated within a canonical 14-3-3 binding motif, showed the highest activity toward the 14-3-3 substrates. These results demonstrate preferential phosphorylation of 14-3-3 proteins by specific CPK paralogs providing a framework for targeted physiological studies. Similar to 14-3-3 proteins, the [alpha]-carboxyltransferase ([alpha]-CT) subunit to the multienzyme complex acetyl-CoA carboxylase (ACCase) was prominently expressed and phosphorylated during seed development. Phosphorylation of [alpha]-CT is noteworthy because the carboxylation of acetyl-CoA to malonyl-CoA by ACCase is the committed step in de novo fatty acid synthesis (FAS). Regulation of ACCase activity by [alpha]-CT phosphorylation therefore, could directly impact the oil composition of seeds. To characterize the role of C-terminal phosphorylation, stable transgenic over-expression lines of Ser to Ala and Ser to Asp (phosphomimetic) single amino acid substitutions were generated (S741A, S741D, S744A, S744D). Fatty acid methyl ester (FAMEs) profiling of homozygous S744D T4 seed revealed a reduced-oil phenotype. Total FA levels of S744D mutant lines averaged 20.3% oil (dry mass). In contrast, empty vector and [alpha]-CT over-expression lines averaged 29.8% and 29.0%, respectively. Specifically, S744D mutant lines were significantly reduced in 16:0, 18:0, 18:1[delta]9, 18:2, and 21:1[delta]11. Reduction in seed oil content, however, did not affect seed mass. These results suggest phosphorylation of the ?-CT C-terminus is of regulatory importance for de novo fatty acid synthesis. The discovery of 14-3-3 specific client proteins, CPK preference for 14-3-3 substrates, and a reduction of seed oil content upon over-expression of phosphomimetic ?-CT subunit to ACCase, all point to the importance of reversible protein phosphorylation during seed development.
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