Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants

Abstract

Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg 2+ as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking.

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“…4). This suggested that ATP hydrolysis in RIL82 plants were adversely inhibited by cold stress, and thus the lower GSH synthesis because of the higher unavailable ATP (Puhakainen et al 1999;Mendoza et al 2000;Deng et al 2015;Muzi et al 2016). This hypothesis was confirmed by the present results that the PARP inhibitor (3-ab) only enhanced the cold tolerance and GSH content in ZZ39 plants under cold stress ( Fig.…”

Salt-Sensitive Signaling Networks in the Mediation of K+/Na+ Homeostasis Gene Expression in Glycyrrhiza uralensis Roots

“…Extracellular ATP levels were monitored with the Enlighten ATP assay system bioluminescence kit (Promega, Madison, WI, United States; Sun et al, 2012a ; Deng et al, 2015 ). Briefly, G. uralensis roots were exposed to 0 or 100 mM NaCl at room temperature.…”

“…eATP was measured in an assay with luciferin-luciferase Turner Designs Modulus TM Microplate Multimode Reader (Promega Corp., Madison, WI, United States). The eATP levels were calculated, based on a standard curve created by measuring a linear range (0.01–100 nM) of standard eATP concentrations ( Sun et al, 2012a ; Deng et al, 2015 ).…”

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