Lee Marie Raytek MSc Candidate, Snedden Lab & Plaxton Lab In vivo hyperphosphorylation of the Ca2+/calmodulin-dependent glutamate decarboxylase isozyme AtGAD1 in phosphate-resupplied Arabidopsis thaliana Inorganic phosphate (Pi) has a crucial role in plant development, yet it is often the most limiting macronutrient of many soils. Pi starved (-Pi) plants elicit a Pi starvation response that alters gene expression and metabolism to enhance their efficiency of Pi acquisition and use. This research field is enabling the development of innovative strategies for engineering Pi-efficient crops, urgently needed to reduce inputs of unsustainable and non-renewable Pi fertilizers for long-term global food security and ecosystem preservation. Our recent phosphoproteomic study revealed that the glutamate decarboxylase AtGAD1 became in vivo phosphorylated at multiple serine residues (located near its N-terminus) 48 h following resupply of 2 mM Pi to -Pi cell cultures of the model plant Arabidopsis thaliana. AtGAD1 is a root-specific, cytosolic GAD isozyme. GADs catalyze the first committed step of the 4-aminobutyrate (GABA) shunt by decarboxylating glutamate into GABA, an important yet enigmatic ‘stress’ metabolite and apparent signal molecule. GAD is the only enzyme of central plant metabolism known to be activated by Ca2+/calmodulin-binding; however the functions or mechanisms of plant GAD phosphorylation have not been studied, although similar N-terminal hyperphosphorylation of AtGAD1 and its orthologs has been described in numerous studies of the phosphoproteome of Arabidopsis and other plants. My thesis research seeks to test two hypotheses: (i) phosphorylation inhibits AtGAD1 activity, and/or (ii) phosphorylation affects AtGAD1’s subcellular localization. This involves comparing the physical and kinetic properties of FPLC-purified, native phospho- versus dephospho-AtGAD1, and molecular cloning of mCherry-AtGAD1 fusion constructs needed to visualize the enzyme’s location in –Pi versus Pi-resupplied cells. Assessing the interplay between Pi nutrition and AtGAD1 phosphorylation will contribute to elucidating the physiological roles of GABA and the GABA shunt under Pi stress and potentially other stresses.
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