Cunfu Lu

NaCl-Induced Alternations of Cellular and Tissue Ion Fluxes in Roots of Salt-Resistant and Salt-Sensitive Poplar Species

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.

H2O2 and cytosolic Ca2+ signals triggered by the PM H-coupled transport system mediate K+/Na+ homeostasis in NaCl-stressed Populus euphratica cells.

Using confocal microscopy, X-ray microanalysis and the scanning ion-selective electrode technique, we investigated the signalling of H(2)O(2), cytosolic Ca(2+) ([Ca(2+)](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. H(2)O(2) levels, regulated upwards by salinity, contributed to ionic homeostasis, because H(2)O(2) restrictions by DPI or DMTU caused enhanced K(+) efflux and decreased Na(+)/H(+) antiport activity. NaCl induced a net Ca(2+) influx and a subsequent rise of [Ca(2+)](cyt), which is involved in H(2)O(2)-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 H(2)O(2) and [Ca(2+)](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 H(2)O(2) and Ca(2+), 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.

Calcium mediates root K+/Na+ homeostasis in poplar species differing in salt tolerance

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.

An ATP signalling pathway in plant cells: extracellular ATP triggers programmed cell death in Populus euphratica

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 H(2) O(2) 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.

Extracellular ATP Signaling Is Mediated by H2O2 and Cytosolic Ca2+ in the Salt Response of Populus euphratica Cells

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.

Ion flux profiles and plant ion homeostasis control under salt stress

The ability of a plant to maintain an ionic homeostasis is crucial in plant salt tolerance. Direct evidence based on data from the non-invasive measurement of ion fluxes would not only offer new insight about the function of the transporter but also provide a whole plant approach for dissecting salt adaptation mechanisms. Here, we review some reports using the ion-selective microelectrodes to characterize the net ion fluxes of tissues or cells.

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

Overexpression of a poplar apyrase gene enhances vesicular trafficking and cold tolerance in Arabidopsis. 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 Mg2+ 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.

Effect of NaCl on leaf H+-ATPase and the relevance to salt tolerance in two contrasting poplar species.

During a 30-day period of increasing salinity, we examined the effects of NaCl on leaf H+-ATPase and salinity tolerance in 1-year-old plants of Populus euphratica Oliv. (salt resistant) and P. popularis 35–44 (P. popularis) (salt sensitive). Electron probe X-ray microanalysis of leaf mesophyll revealed that P. euphratica had a higher ability to retain lower NaCl concentrations in the cytoplasm, as compared to P. popularis. The sustained activity of H+ pumps (by cytochemical staining) in salinised P. euphratica suggests a role in energising salt transport through the plasma membrane (PM) and tonoplast. Salt-induced alterations of leaf respiration, ATP content and expression of PM H+-ATPase were compared between the two species. Results show that P. euphratica retained a constant respiratory rate, ATP production and protein abundance of PM H+-ATPase (by Western blotting) in salt-stressed plants. P. euphratica was able to maintain a comparatively high capacity of ATP hydrolysis and H+ pumping during prolonged salt exposure. By contrast, the activity and expression of PM H+-ATPase were markedly decreased in P. popularis leaves in response to salt stress. Furthermore, NaCl-stressed P. popularis plants showed a marked decline of respiration (70%) and ATP production (66%) on day 30. We conclude that the inability of P. popularis to transport salt to the apoplast and vacuole was partly due to the decreased activity of H+ pumps. As a consequence, cytosolic ion concentrations were observed to be comparatively high for an extended period of time, so that cell metabolism, in particular respiration, was disrupted in P. popularis leaves.

Hydrogen Sulfide Mediates K+ and Na+ Homeostasis in the Roots of Salt-Resistant and Salt-Sensitive Poplar Species Subjected to NaCl Stress

Non-invasive micro-test techniques (NMT) were used to analyze NaCl-altered flux profiles of K+, Na+, and H+ in roots and effects of NaHS (a H2S donor) on root ion fluxes in two contrasting poplar species, Populus euphratica (salt-resistant) and Populus popularis (salt-sensitive). Both poplar species displayed a net K+ efflux after exposure to salt shock (100 mM NaCl), as well as after short-term (24 h), and long-term (LT) (5 days) saline treatment (50 mM NaCl, referred to as salt stress). NaHS (50 μM) restricted NaCl-induced K+ efflux in roots irrespective of the duration of salt exposure, but K+ efflux was not pronounced in data collected from the LT salt stress treatment of P. euphratica. The NaCl-induced K+ efflux was inhibited by a K+ channel blocker, tetraethylammonium chloride (TEA) in P. popularis root samples, but K+ loss increased with a specific inhibitor of plasma membrane (PM) H+-ATPase, sodium orthovanadate, in both poplar species under LT salt stress and NaHS treatment. This indicates that NaCl-induced K+ loss was through depolarization-activated K+ channels. NaHS caused increased Na+ efflux and a corresponding increase in H+ influx for poplar roots subjected to both the short- and LT salt stress. The NaHS-enhanced H+ influx was not significant in P. euphratica samples subjected to short term salt stress. Both sodium orthovanadate and amiloride (a Na+/H+ antiporter inhibitor) effectively inhibited the NaHS-augmented Na+ efflux, indicating that the H2S-enhanced Na+ efflux was due to active Na+ exclusion across the PM. We therefore conclude that the beneficial effects of H2S probably arise from upward regulation of the Na+/H+ antiport system (H+ pumps and Na+/H+ antiporters), which promote exchange of Na+ with H+ across the PM and simultaneously restricted the channel-mediated K+ loss that activated by membrane depolarization.