Populus Euphratica Apyrase2 Enhances Cold Tolerance By Modulating Vesicular Trafficking And Extracellular ATP In Arabidopsis Plants

Low temperature is a major environmental factor that restrains plant growth and crop productivity. When temperatures fall below 0℃, plant cells experience dehydration and mechanical injury caused by ice crystallization. Cold stress reduces plasma membrane (PM) integrity, which leads to leakage of intracellular solutes. ATP can be an important signaling molecule when released into the extracellular matrix (ECM). Extracellular ATP (eATP) was shown to regulate a wide range of cellular processes, but its functions are dose-dependent. In addition, eATP also plays a fundamental role in mediating plant responses to environmental stresses, such as pathogens, wounds, high salt, and osmotic stress. However, the link between eATP and cold tolerance has not been fully established. Ecto Nucleoside Triphosphate Diphosphohydrolases (Ecto-apyrases) are the principal enzymes that limit eATP accumulation in both animals and plants. In Arabidopsis, apyrases are suggested to be involved in some of the signaling steps in plant growth and defense response, including pollen germination, cotton fiber elongation, root hair growth, and stomatal movement. Apyrase was also postulated to serve as a signal in stress responses. However, no studies have investigated in higher-order plants whether apyrase promotes hydrolysis of ATP at low temperatures and whether this activity is correlated to low temperature tolerance.This study evaluated the roles of apyrase and eATP in cold stress signaling in woody plants. We focused on P. euphratica, because this species plays very important roles in stabilizing sand dunes and in sheltering agricultural regions in north-west China. In addition, P. euphratica trees can adapt to harsh temperature conditions in saline and alkaline desert sites. In the present study, we showed that cold stress induced release of ATP due to the impaired plasma membrane in P. euphratica callus cells. In correlation to the increased eATP level, expression of APY2 was up-regulated in P. euphratica cells. While the expression of another apyrase PeAPYl did not induced by cold. Thus, APY2 may contribute to cold adaptation in P. euphratica. We tested this hypothesis by cloning the PeAPY2 gene from P. euphratica callus cells, and characterized its function under low temperature stress.1. Using transient expression of YFP-tagged PeAPY2 in anion epidermal cells and Aradidopsis protoplasts, we found that PeAPY2 predominantly localized to the plasma membrane, but punctuate signals also appeared in the endoplasmic reticulum and Golgi apparatus. The results suggested that PeAPY2 may be sorted into plasma membrane through secretory pathway.2. Enzymatic activity analysis of purified PeAPY2 showed that PeAPY2 exhibited broad substrate specificity, including purine nucleotides (ATP and GTP), pyrimidine nucleotides (CTP and UTP) or diphosphate nucleotides (e.g. ADP and UTP). But it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 apyrase activity increased with divalent ion cofactors, particularly Mg2+. Moreover, PeAPY2 is insensitive to specific inhibitors of P-, V-, and F-type ATPases and various types of phosphatases.3. We investigated the roles of PeAPY2 in eATP control and low temperature tolerance by transferring it into a model species, Arabidopsis thaliana. When PeAPY2 was ectopically expressed in Arabidopsis, cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under low temperature stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared to wild type plants, suggesting that PeAPY2 enhanced plasma membrane repair in transgenic plants.4. By analyzing the expression of a series of genes widely involved in stress and membrane resealing, we found that PeAPY2 transgenic plants showed marked up-regulation of the synaptotagmin gene, AtSYT1, which contributes to plasma membrane repair under freezing stress.5. Of note, PeAPY2 transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. Moreover, 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. Thus, the potential suppression of vesicular trafficking by high eATP was avoided in PeAPY2-transgenic plants during cold stress and recovery.In conclusion, cold treatment inhibits vesicular trafficking and increases electrolyte leakage in the plasma membrane, which results in ATP release to the ECM. The excess eATP inhibits vesicular trafficking and membrane repair, which leads to membrane disruption and decreased cell viability. When Arabidopsis plants overexpress PeAPY2, they show enhanced vesicular trafficking and highly active ATP hydrolysis, which then reduces the excess accumulation of cold-elicited eATP in the ECM. Thus, PeAPY2 protects membrane integrity, which benefits transgenic plant growth and survival during cold stress and recovery. In P. euphratica cells, PeAPY2 was upregulated under low-temperature conditions. The activated PeAPY2 enhanced vesicular trafficking and modulated eATP levels to reduce the ATP inhibition of vesicular trafficking and membrane repair. Consequently, the ability to tolerate low temperature was improved in P. euphratica.