A radiolabeling approach to purinoceptor-like receptor identification in plants and evidence for apyrase (APY1 and APY2) regulation of stomatal aperture in arabidopsis



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Adenosine triphosphate (ATP) is well recognized for its role as the primary cellular energy currency. However, studies dating back to 1929 have reshaped our understanding of ATP as not only an energy source, but also as a signaling agent. Among the most important of these discoveries are animal purinergic receptors (P2X and P2Y receptors) that perceive extracellular ATP (eATP), primarily in the nervous system. Though eATP is an established receptor agonist in animals and applied poorly hydrolyzable ATP analogs have numerous effects on growth in plants, eATP is not widely accepted as a signal in plants where no purinoceptor has been identified. Here, enriched outside-out plasma membrane vesicles were isolated and proteins labeled with a radioactive ATP analog (8N₃ATP[α²³P]) to identify a putative purinoceptor-like receptor. We used etiolated seedlings to capture proteins from plant tissue that was actively growing and used sodium carbonate washes to separate peripheral and integral membrane proteins. With this method, we have generated lists of plasma membrane ATP binding proteins, and therefore possible eATP receptors. Ectoapyrases are phosphohydrolases thought to regulate eATP in both animals and plants. Here, we also investigated the expression and role of the candidate ectoapyrases AtAPY1 and AtAPY2 in guard cells and stomatal responses. AtAPY1 and AtAPY2 transcript and protein expression was confirmed in guard cells. Early genetic studies using an apy2 knock out with induced RNAi-silencing of APY1 suggest a role for these apyrases in stomatal regulation. In response to treatment with five hours light, the apyrase-suppressed line features wider stomatal aperture when compared to WS wild-type.