Using the scanning ion-selective electrode technique, fluxes of H+, Na+, and Cl− were investigated in roots and derived protoplasts of salt-tolerant Populus euphratica and salt-sensitive Populus popularis 35-44 (P. popularis). Compared to P. popularis, P. euphratica roots exhibited a higher capacity to extrude Na+ after a short-term exposure to 50 mm NaCl (24 h) and a long term in a saline environment of 100 mm NaCl (15 d). Root protoplasts, isolated from the long-term-stressed P. euphratica roots, had an enhanced Na+ efflux and a correspondingly increased H+ influx, especially at an acidic pH of 5.5. However, the NaCl-induced Na+/H+ exchange in root tissues and cells was inhibited by amiloride (a Na+/H+ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H+-ATPase inhibitor). These results indicate that the Na+ extrusion in stressed P. euphratica roots is the result of an active Na+/H+ antiport across the plasma membrane. In comparison, the Na+/H+ antiport system in salt-stressed P. popularis roots was insufficient to exclude Na+ at both the tissue and cellular levels. Moreover, salt-treated P. euphratica roots retained a higher capacity for Cl− exclusion than P. popularis, especially during a long term in high salinity. The pattern of NaCl-induced fluxes of H+, Na+, and Cl− differs from that caused by isomotic mannitol in P. euphratica roots, suggesting that NaCl-induced alternations of root ion fluxes are mainly the result of ion-specific effects.

Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H2O2, cytosolic Ca2+ ([Ca2+]cyt) and the PM H+‐coupled transport system in K+/Na+ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na+/H+ antiport was seen in salinized cells; however, NaCl stress caused a net K+ efflux, because of the salt‐induced membrane depolarization. H2O2 levels, regulated upwards by salinity, contributed to ionic homeostasis, because H2O2 restrictions by DPI or DMTU caused enhanced K+ efflux and decreased Na+/H+ antiport activity. NaCl induced a net Ca2+ influx and a subsequent rise of [Ca2+]cyt, which is involved in H2O2‐mediated K+/Na+ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na+/H+ antiport system, the NaCl‐induced elevation of H2O2 and [Ca2+]cyt was correspondingly restricted, leading to a greater K+ efflux and a more pronounced reduction in Na+/H+ antiport activity. Results suggest that the PM H+‐coupled transport system mediates H+ translocation and triggers the stress signalling of H2O2 and Ca2+, which results in a K+/Na+ homeostasis via mediations of K+ channels and the Na+/H+ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.

Using the non-invasively ion-selective microelectrode technique, flux profiles of K(+), Na(+) and H(+) in mature roots and apical regions, and the effects of Ca(2+) on ion fluxes were investigated in salt-tolerant poplar species, Populus euphratica Oliver and salt-sensitive Populus simonii x (P. pyramidalis + Salix matsudana) (Populus popularis 35-44, P. popularis). Compared to P. popularis, P. euphratica roots exhibited a greater capacity to retain K(+) after exposure to a salt shock (SS, 100 mM NaCl) and a long-term (LT) salinity (50 mM NaCl, 3 weeks). Salt shock-induced K(+) efflux in the two species was markedly restricted by K(+) channel blocker, tetraethylammonium chloride, but enhanced by sodium orthovanadate, the inhibitor of plasma membrane (PM) H(+)-ATPase, suggesting that the K(+) efflux is mediated by depolarization-activated (DA) channels, e.g., KORCs (outward rectifying K(+) channels) and NSCCs (non-selective cation channels). Populus euphratica roots were more effective to exclude Na(+) than P. popularis in an LT experiment, resulting from the Na(+)/H(+) antiport across the PM. Moreover, pharmacological evidence implies that the greater ability to control K(+)/Na(+) homeostasis in salinized P. euphratica roots is associated with the higher H(+)-pumping activity, which provides an electrochemical H(+) gradient for Na(+)/H(+) exchange and simultaneously decreases the NaCl-induced depolarization of PM, thus reducing Na(+) influx via NSCCs and K(+) efflux through DA-KORCs and DA-NSCCs. Ca(2+) application markedly limited salt-induced K(+) efflux but enhanced the apparent Na(+) efflux, thus enabling the two species, especially the salt-sensitive poplar, to retain K(+)/Na(+) homeostasis in roots exposed to prolonged NaCl treatment.

Using callus cells of a salt-tolerant Populus euphratica Oliver and a salt-sensitive P. popularis 35-44 (P. popularis), the effects of NaCl stress on hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) production and the relevance to ionic homeostasis and antioxidant defense were investigated. Results show that P. euphratica exhibited a greater capacity to tolerate NaCl stress in terms of cell viability, membrane permeability and K ? /Na ? relations. NaCl salinity (150 mM) caused a rapid increase of H 2 O 2 and NO in P. euphratica cells, but not in P. popularis. Moreover, salinised P. euphratica cells retained a high and stable level of H 2 O 2 and NO during the period of 24-h salt stress. Noteworthy, P. eupratica cells increased activities of superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase under salinity stress, but these antioxidant enzymes were significantly inhibited by the salt treatment in P. popularis cells. Pharmacological experiments proved that the NaCl-induced H 2 O 2 and NO was interdependent and contributed to the mediation of K ? /Na ? homeostasis and antioxidant defense in P. euphratica cells. Given these results, we conclude that the increased H 2 O 2 and NO enable P. euphratica cells to regulate ionic and ROS (reactive oxygen species) homeostasis under salinity stress in the longer term.

Extracellular ATP (eATP) has been implicated in mediating plant growth and antioxidant defense; however, it is largely unknown whether eATP might mediate salinity tolerance. We used confocal microscopy, a non-invasive vibrating ion-selective microelectrode, and quantitative real time PCR analysis to evaluate the physiological significance of eATP in the salt resistance of cell cultures derived from a salt-tolerant woody species, Populus euphratica. Application of NaCl (200 mM) shock induced a transient elevation in [eATP]. We investigated the effects of eATP by blocking P2 receptors with suramin and PPADS and applying an ATP trap system of hexokinase-glucose. We found that eATP regulated a wide range of cellular processes required for salt adaptation, including vacuolar Na+ compartmentation, Na+/H+ exchange across the plasma membrane (PM), K+ homeostasis, reactive oxygen species regulation, and salt-responsive expression of genes related to K+/Na+ homeostasis and PM repair. Furthermore, we found that the eATP signaling was mediated by H2O2 and cytosolic Ca2+ released in response to high salt in P. euphratica cells. We concluded that salt-induced eATP was sensed by purinoceptors in the PM, and this led to the induction of downstream signals, like H2O2 and cytosolic Ca2+, which are required for the up-regulation of genes linked to K+/Na+ homeostasis and PM repair. Consequently, the viability of P. euphratica cells was maintained during a prolonged period of salt stress.

We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose-and time-dependent reduction in viability, and the agonist-treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca 2+ levels, resulting in Ca 2+ uptake by the mitochondria and subsequent H2O2 accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP-induced increase of intracellular ATP, essential for the activation of caspase-like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca 2+ concentration but plays a negligible role in eATP-stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.

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.

Using the Affymetrix poplar genome array, we explored the leaf transcriptome of salt-tolerant Populus euphratica Oliv. and salt-sensitive P. popularis 35-44 (P. popularis) under control and saline conditions. Our objective was to clarify the genomic differences in regulating K(+)/Na(+) and reactive oxygen species (ROS) homeostasis between the two species. Compared to P. popularis, salt-tolerant P. euphratica responses to salinity involved induction of a relatively larger number of probesets after short-term (ST) exposure to 150 mM NaCl (24 h) and relatively fewer probesets after a long-term (LT) exposure to salinity (200 mM NaCl, 28 days). Compared to P. popularis, leaves of the control P. euphratica plants exhibited a higher transcript abundance of genes related to Na(+)/H(+) antiport (Na(+)/H(+) antiporters, H(+) pumps) and K(+) uptake and transport. Notably, the expression of these genes did not decrease (with a few exceptions) during salt treatment. Regarding ROS homeostasis, P. euphratica exhibited rapid up-regulation of a variety of antioxidant enzymes after exposure to ST salinity, indicating a rapid adaptive response to salt stress. However, the effect of NaCl on transcription in P. popularis leaves was more pronounced after exposure to prolonged salinity. LT-stressed P. popularis up-regulated some genes mediating K(+)/Na(+) homeostasis but decreased transcription of main scavengers of superoxide radicals and H(2)O(2) except for some isoforms of a few scavengers. Mineral and ROS analyses show that NaCl induced a marked increase of leaf Na(+) and H(2)O(2) in LT-stressed plants of the two species and the effects were even more pronounced in the salt-sensitive poplar. We place the transcription results in the context of our physiological measurements to infer some implications of NaCl-induced alterations in gene expression related to K(+)/Na(+) and ROS homeostasis.