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.

Figure 1.

Effects of cold stress on eATP, electrolyte leakage (EL), malondialdehyde (MDA) content, and expression profiles of PeAPY2 in P. euphratica callus cells. P. euphratica cells were subjected to 4°C for low-temperature treatment, while control cells were cultured under normal growth conditions at 25°C. A, eATP, electrolyte leakage, and malondialdehyde under cold stress. Cold-stressed and control cells were daily sampled during the period of cold stress. FW, Fresh weight. B, PeAPY2 mRNA levels during cold stress. Cold-stressed and control cells were sampled after 0, 1, 5, 8, 11, 24, 48, 72, and 168 h of treatment. The expression levels of PeAPY2 were normalized to the expression level of the P. euphratica β-ACTIN7 gene (PeACT7; internal control) to derive relative expression. Primers designed to target PeAPY2 and PeACT7 genes are shown in Supplemental Table S3. Each point is the mean of three independent experiments, and error bars represent se. *, P < 0.05, **, P < 0.01, control versus cold treatment.

Figure 2.

Subcellular localization of PeAPY2 after transient transformation in onion epidermal cells and Arabidopsis mesophyll protoplasts. A, Diagrams of the eYFP control (top) and PeAPY2-eYFP fusion (bottom) constructs for transformation. B, Representative images of PeAPY2-eYFP transgenic onion cells and eYFP controls. Plasmolysis was induced by hyperosmotic shock with 500 mm NaCl. CW, Cell wall; HS, Hechtian strands; N, nucleus. Bars = 50 μm. C, Representative images show the colocalization (yellow-green in merged images) of PeAPY2-eYFP (yellow) and CFP-tagged (cyan) organelle markers for the PM (PM-CFP; Arabidopsis Biological Resource Center [ABRC] stock no. CD3-1001), ER (ER-CFP; ABRC stock no. CD3-953), and Golgi apparatus (Golgi-CFP; ABRC stock no. CD3-961) in Arabidopsis mesophyll protoplasts. Bars = 5 μm.

Figure 3.

Enzymatic characterization of PeAPY2 recombinant protein. A, Substrate specificity of PeAPY2. Nucleotide triphosphates (ATP, GTP, CTP, and UTP), nucleotide diphosphates (ADP and UDP), and AMP nucleotides (AMP) were applied at a concentration of 3 mm. B, Sensitivity of PeAPY2 to specific inhibitors of various ATPases. The ATPase inhibitors were applied at different concentrations: NaF (fluoride, an inhibitor of pyrophosphatases; 40 mm), NaN₃ (N₃, an inhibitor of F-type ATPases; 5 mm), NaNO₃ (NO₃, an inhibitor of V-type ATPases; 50 mm), Na₃VO₄ (VO₄, an inhibitor of P-type ATPases; 1 mm), NaMoO₄ (molybdate, an inhibitor of acid phosphatases; 1 mm), and NGXT191 (an inhibitor of apyrases from Arabidopsis and potato; 5 μg mL⁻¹). PeAPY2 activities are expressed relative to the control activity in the absence of inhibitors. C, Effects of different divalent metal ions on PeAPY2 apyrase activity. Apyrase activity was measured in the absence (control) and presence of divalent metal ions (Mg²⁺, Ca²⁺, Zn²⁺, Cd²⁺, Mn²⁺, Co²⁺, Cu²⁺, and Ni²⁺; 3 mm) and a 1:1 combination of Mg²⁺ and Ca²⁺ (1.5 mm Mg²⁺ + 1.5 mm Ca²⁺). EDTA (3 mm) was applied to chelate divalent ions of Mg²⁺ and/or Ca²⁺. Each column is the mean of three independent experiments, and error bars represent se. Columns with different letters showed significant differences, with P < 0.05.

Figure 4.

Cold tolerance of wild-type (WT), vector control (VC), and PeAPY2-transgenic (OE) Arabidopsis lines. A, qRT-PCR analysis results show the relative expression of PeAPY2 compared with the expression of the Arabidopsis housekeeping gene, ACTIN2. Columns labeled with asterisks denote significant differences at P < 0.05 between transgenic lines. B and C, Freeze tolerance tests show responses of wild-type and transgenic plants. Seeds from wild-type, vector control, and transgenic lines (OE2 and OE4; T3 generation) were allowed to germinate on one-half-strength MS medium. Seven-day-old seedlings of all tested lines were acclimated at 4°C for 1 week and then transferred to a programmed freezing chamber. Petri dishes were maintained at −1°C for 6 h, and then the temperature was lowered to −6°C at a rate of 1°C h⁻¹. After 2 h of freezing treatment, the plates were removed to 4°C in the dark for 12 h and then transferred to 22°C under light for recovery. The survival rates of cold-stressed plants were measured after 10 d of recovery. Control plants were grown at 22°C in a long-day light period. Representative images (B) show plant performance before (top) and after (bottom) cold stress at −6°C. Survival rates (C) after recovery from freezing (−6°C) were compared with controls maintained at 22°C (100% survival). Each column is the mean of three independent experiments, and error bars represent se. *, P < 0.05 for the wild type and vector controls versus transgenic lines after cold and recovery treatment.

Figure 5.

Effects of cold stress on root length, cell viability, and electrolyte leakage in wild-type (WT), vector control (VC), and PeAPY2-transgenic (OE) Arabidopsis lines. A, Cell viability. T3 seeds of wild-type, vector control, and PeAPY2-transgenic (OE2 and OE4) lines were germinated on one-half-strength MS medium. Seven-day-old seedlings of all tested lines were cold acclimated for 7 d and freezing treated at −1°C for 12 h. Thereafter, the seedlings were removed to 4°C for 12 h. Control plants were grown at 22°C in a long-day light period. Cell viability was assayed with fluorescein diacetate (FDA; green) and FM4-64 (red) double staining; representative images of the elongation zone of roots are shown. The ratio of FM4-64 to FDA fluorescence was calculated, and values were normalized to the wild-type control at 22°C. Values ± sd labeled with different letters showed significant differences at P < 0.05. Bars = 30 μm. B, Root length. Seven-day-old seedlings of wild-type, vector control, and PeAPY2-transgenic lines (T3 generation) were maintained at 4°C for 1 week. Root lengths of plants exposed to cold stress (4°C for 7 d) are expressed relative to non-cold-stressed controls (100%) of the same strains. C, Electrolyte leakage. Seven-day-old seedlings of all tested lines were acclimated at 4°C for 1 week and then subjected to an increasing freezing stress. Seedlings were exposed to −1°C for 6 h, and then the temperature was lowered to −6°C at a rate of 1°C h⁻¹. After 2 h of −6°C treatment, plants were removed to 4°C in the dark for 12 h and then transfer to 22°C under light for recovery. Electrolyte leakage of cold-stressed plants was measured after 3 d of recovery. Control plants were grown at 22°C in a long-day light period. In B and C, each column is the mean of three independent experiments, and error bars represent se. Columns labeled with different letters showed significant differences at P < 0.05.

Figure 6.

Effects of apyrase inhibitors and brefeldin A (BFA) on vesicular trafficking in root cells of wild-type (WT) and PeAPY2-transgenic plant lines. T3 seeds of wild-type and PeAPY2-transgenic lines (OE2 and OE4) were germinated on one-half-strength MS medium, and 7-d-old seedlings of all tested lines were used for FM4-64 staining (red). A and B, Endocytosis. Seedlings were stained with FM4-64 for 10 min and then washed for 15 min (A) or 30 min (B). Endocytic vesicles are stained red. C, Apyrase inhibitor treatment. Prior to FM4-64 staining, wild-type roots were treated with NGXT191 (3 μg mL⁻¹), and PeAPY2-transgenic lines were treated with polyclonal antibodies against PeAPY2 at a dilution of 1:500, for 1.5 h. The uptake of FM4-64 was calculated as the ratio of intracellular fluorescence to whole-cell fluorescence, and values were standardized to the wild-type control with 30 min of washing of FM4-64. In A to C, values ± sd labeled with different letters showed significant differences at P < 0.05. D and E, Exocytosis. After FM4-64 staining, wild-type and transgenic plant roots were incubated with BFA (50 μm for 1.5 h). Images show FM4-64 uptake before (D) and after (E) washing out BFA for 1.5 h. Arrowheads indicate BFA bodies. BFA bodies were quantified as the ratio of intracellular surface to the whole cell, and values were standardized to the wild-type control before BFA washing. Values ± sd labeled with different letters showed significant differences at P < 0.05. Bars = 10 μm.

Figure 7.

Effects of low temperatures on vesicular trafficking in root cells of wild-type (WT) and PeAPY2-transgenic lines. A and B, T3 seeds of wild-type and PeAPY2-transgenic lines (OE2 and OE4) were germinated on one-half-strength MS medium. Seven-day-old plants were cultured under control growth conditions at 22°C (A) or exposed to 4°C for 24 h (B). Control and cold-stressed roots were stained with FM4-64 for 10 min, followed by a 30-min washing. The uptake of FM4-64 was calculated as the ratio of intracellular fluorescence to whole-cell fluorescence, and values were standardized to the wild-type control. Values ± sd labeled with different letters showed significant differences at P < 0.05. C and D, Seven-day-old seedlings were cold acclimated (4°C for 7 d) and then exposed to −1°C for 6 h. Next, the seedlings were moved to 4°C for 12 h in the dark and then allowed to recover at 22°C for 1 d. After FM4-64 staining, roots of wild-type and transgenic lines were incubated with BFA (50 μm for 1.5 h). Images show FM4-64 uptake before (C) and after (D) washing out BFA for 1.5 h. Arrowheads indicate BFA bodies. BFA bodies were quantified as the ratio of intracellular surface to the whole cell, and values were standardized to the wild-type control before BFA washing. Values ± sd labeled with different letters showed significant differences at P < 0.05. Bars = 10 μm.

Figure 8.

Effects of cold stress on eATP concentrations and eATP inhibition of cell viability in wild-type (WT) and PeAPY2-transgenic lines. T3 seeds of wild-type and PeAPY2-transgenic lines (OE2 and OE4) were germinated on one-half-strength MS medium. A, Seven-day-old seedlings of all tested lines were subjected to 4°C for 7 d (cold stress), followed by −1°C for 12 h and 4°C for 12 h, and were finally returned to 22°C for 12 h of recovery (R12h). [eATP] was measured in extracellular root medium. Each point is the mean of three independent experiments, and error bars represent se. **, P < 0.01, wild-type versus PeAPY2-transgenic lines. B, eATP inhibition of cell viability. Seven-day-old seedlings of wild-type and PeAPY2-transgenic lines (OE2 and OE4) were treated with 0 or 800 μm ATP for 12 h. Equal molar values of CaCl₂ were applied for the ATP treatment, and pH was adjusted to 5.7 to 5.8 when ATP was added into the nutrient solution. Root cell viability was assayed with FDA (green) and PI (red) staining. The merged images show the double staining. Three independent experiments were performed, and representative images are shown. Bars = 250 μm.

Figure 9.

Effects of ATP on viability in root cells of wild-type (WT) and PeAPY2-transgenic lines under cold stress. T3 seeds of wild-type and PeAPY2-transgenic lines (OE2 and OE4) were germinated on one-half-strength MS medium. Roots of wild-type and transgenic seedlings (7 d old) were exposed to 0, 50, or 600 μm ATP for 24 h. Equal molar values of CaCl₂ were applied for the ATP treatment, and pH was adjusted to 5.7 to 5.8 when ATP was added into the nutrient solution. These seedlings of wild-type and transgenic lines were cold acclimated at 4°C for 1 d and subjected to low-temperature treatment: −1°C for 16 h, then the temperature was lowered to −6°C for 2 h and finally recovered at 4°C for 12 h. Control plants were grown at 22°C in a long-day light period. Then, roots were stained with FDA (green) and PI (red) to examine root cell viability with a confocal microscope. Three independent experiments were performed, and representative images are shown. Bar = 10 μm.

Figure 10.

Effects of hydrolyzable ATP and nonhydrolyzable ATPγS on vesicular trafficking in root cells of wild-type (WT) and PeAPY2-transgenic lines. T3 seeds of wild-type and PeAPY2-transgenic lines (OE2 and OE4) were germinated on one-half-strength MS medium. Seven-day-old seedlings were exposed to different concentrations of eATP (0, 50, 500, and 1,000 μm) or extracellular ATPγS (500 μm) for 1.5 h. Equal molar values of CaCl₂ were applied for the ATP treatment, and pH was adjusted to 5.7 to 5.8 when ATP was added into the nutrient solution. Then, roots were stained with FM4-64 for 10 min and washed for 30 min. Three independent experiments were performed, and representative images are shown. The uptake of FM4-64 was calculated as the ratio of intracellular fluorescence to whole-cell fluorescence, and values were standardized to the wild-type control treated without ATP or ATPγS. Values ± sd labeled with different letters showed significant differences at P < 0.05. Bars = 10 μm.

Figure 11.

Schematic model showing the role of P. euphratica apyrase, PeAPY2, in cold tolerance and recovery from cold stress.