What is the role of phosphorylation of the Ca2+/calmodulin-dependent glutamate decarboxylase isozyme, AtGAD1, in response to phosphate nutrition of Arabidopsis thaliana?

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Raytek, Lee
Biochemistry , GABA , Plants , phosphate
Orthophosphate (H2PO4−; Pi) is an essential but limiting macronutrient required for many fundamental aspects of plant metabolism, particularly photosynthesis and respiration. Pi starved (-Pi) plants elicit a Pi starvation response that alters gene expression and metabolism to enhance their efficiency of Pi acquisition and use. We recently employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess the impact of Pi nutrition on the phosphoproteome of cell cultures of the model plant Arabidopsis thaliana. Forty-eight hours following Pi resupply to -Pi cells, the glutamate decarboxylase isozyme AtGAD1 (AT5G17330.1) became in vivo hyperphosphorylated at multiple seryl residues near its N-terminus. AtGAD1 is a root-specific, Ca2+/calmodulin-activated cytosolic isozyme that catalyzes the first committed step of the γ-aminobutyrate (GABA) shunt by decarboxylating glutamate into GABA. Although phosphoproteomic studies have documented widespread plant GAD phosphorylation, there have been no reports on the functions nor mechanisms of plant GAD phosphorylation. Here, I developed and validated a spectrophotometric GAD activity assay, as well as an anti-(pSer8 phosphosite-specific) antibody (α-pSer8) to corroborate our LC-MS/MS detection of AtGAD1 phosphorylation. I fully purified AtGAD1 from Pi-sufficient (+Pi) and -Pi cells (+P:GAD1 and -P:GAD1, respectively) to final specific activities of 20 and 32 (μmol GABA produced/min)mg-1, respectively. LC-MS/MS, SDS-PAGE, immunoblotting, and analytical gel-filtration indicated that both final preparations exist as a homohexamer composed of identical 57 kDa AtGAD1 subunits. The 60% higher specific activity of purified -P:GAD1 relative to +P:GAD1 correlated with their differential phosphorylation as detected by LC-MS/MS and α-pSer8 immunoblotting, wherein -P:GAD1 was more highly phosphorylated at Ser4, Ser10, and Ser13, but was hypophosphorylated at Ser8 compared to +P:GAD1. Imaging fluorescent-protein tagged AtGAD1 in Arabidopsis suspension cells, as well as microsomal fractionation and immunoblotting, indicated that GAD’s cytosolic localization was unaffected by changes in nutritional Pi status. Collectively, my data suggest that Pi starvation increases the specific activity of GAD through differential phosphorylation. This change is expected to lead to greater GABA accumulation or flux through the GABA shunt in -Pi plants. This work adds to the literature showing that the shunt is important during abiotic stress responses and expands the knowledge base for future engineering of improved Pi-use efficiency in crops.
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