Shaoliang Chen

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.

Sodium and chloride distribution in roots and transport in three poplar genotypes under increasing NaCl stress

Abstract We investigated the effects of increasing soil NaCl on growth, water flow, ion transport and intracellular compartmentation of Na + and Cl − in 1-year-old seedlings of Populus euphratica Oliv. and 1-year-old rooted cuttings of P .× euramericana cv. I-214 (cv. Italica) and P. ‘ popularis 35-44’ ( P. popularis ). Relative growth rates of leaf area (RGR A ) and shoot height (RGR H ) of cv. Italica and P. popularis were severely restricted by increasing salinity, whereas in P. euphratica both were not significantly inhibited . Salinised trees of P. euphratica experienced 10% leaf area loss during a 30-day study, however, cv. Italica and P. popularis shed over 50% of their initial surface area. Leaf necrosis of the two salt-sensitive genotypes (cv. Italica and P. popularis ) was attributed to excessive salt accumulation and reduced water loss. The rapid built-up of leaf salt in these two genotypes was mainly the result of high ion concentrations in the transpiration stream. An artificially generated lower shoot-to-root ratio, which was reached by removing approximately 50% of total leaf area from shoots of cv. Italica and P. popularis prior to the salt treatment did not enhance their salinity tolerance since root-to-shoot salt fluxes were largely independent of water flow. Compared with cv. Italica and P. popularis , P. euphratica maintained considerably higher unit leaf transpiration rates with lower salt concentrations in the transpiration stream during the period of salt stress. Therefore, salt tolerance of P. euphratica likely depends on its ability to restrict salt transport to leaves. X-ray microanalysis of root compartments showed that there was genotypic difference in the pattern of ion compartmentation. Cv. Italica exhibited a greater capacity to accumulate salt in cortical vacuoles compared with P. popularis , even though both were considered as salt-sensitive genotypes. P. euphratica was more effective than the other two genotypes to block apoplasmic ion transport and sequester Cl − in cortical vacuoles at high salinity. These limited ion loading into the xylem during radial transport, and thus contributed to the restriction of subsequent axial transport.

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.

On the salty side of life: molecular, physiological and anatomical adaptation and acclimation of trees to extreme habitats

Saline and sodic soils that cannot be used for agriculture occur worldwide. Cultivating stress-tolerant trees to obtain biomass from salinized areas has been suggested. Various tree species of economic importance for fruit, fibre and timber production exhibit high salinity tolerance. Little is known about the mechanisms enabling tree crops to cope with high salinity for extended periods. Here, the molecular, physiological and anatomical adjustments underlying salt tolerance in glycophytic and halophytic model tree species, such as Populus euphratica in terrestrial habitats, and mangrove species along coastlines are reviewed. Key mechanisms that have been identified as mediating salt tolerance are discussed at scales from the genetic to the morphological level, including leaf succulence and structural adjustments of wood anatomy. The genetic and transcriptomic bases for physiological salt acclimation are salt sensing and signalling networks that activate target genes; the target genes keep reactive oxygen species under control, maintain the ion balance and restore water status. Evolutionary adaptation includes gene duplication in these pathways. Strategies for and limitations to tree improvement, particularly transgenic approaches for increasing salt tolerance by transforming trees with single and multiple candidate genes, are discussed.

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.

Populus euphratica XTH overexpression enhances salinity tolerance by the development of leaf succulence in transgenic tobacco plants

Populus euphratica is a salt-tolerant tree species that develops leaf succulence after a prolonged period of salinity stress. In the present study, a putative xyloglucan endotransglucosylase/hydrolase gene (PeXTH) from P. euphratica was isolated and transferred to tobacco plants. PeXTH localized exclusively to the endoplasmic reticulum and cell wall. Plants overexpressing PeXTH were more salt tolerant than wild-type tobacco with respect to root and leaf growth, and survival. The increased capacity for salt tolerance was due mainly to the anatomical and physiological alterations caused by PeXTH overexpression. Compared with the wild type, PeXTH-transgenic plants contained 36% higher water content per unit area and 39% higher ratio of fresh weight to dry weight, a hallmark of leaf succulence. However, the increased water storage in the leaves in PeXTH-transgenic plants was not accompanied by greater leaf thickness but was due to highly packed palisade parenchyma cells and fewer intercellular air spaces between mesophyll cells. In addition to the salt dilution effect in response to NaCl, these anatomical changes increased leaf water-retaining capacity, which lowered the increase of salt concentration in the succulent tissues and mesophyll cells. Moreover, the increased number of mesophyll cells reduced the intercellular air space, which improved carbon economy and resulted in a 47–78% greater net photosynthesis under control and salt treatments (100–150mM NaCl). Taken together, the results indicate that PeXTH overexpression enhanced salt tolerance by the development of succulent leaves in tobacco plants without swelling.

Paxillus involutus Strains MAJ and NAU Mediate K+/Na+ Homeostasis in Ectomycorrhizal Populus × canescens under Sodium Chloride Stress

Salt-induced fluxes of H+, Na+, K+, and Ca2+ were investigated in ectomycorrhizal (EM) associations formed by Paxillus involutus (strains MAJ and NAU) with the salt-sensitive poplar hybrid Populus × canescens. A scanning ion-selective electrode technique was used to measure flux profiles in non-EM roots and axenically grown EM cultures of the two P. involutus isolates to identify whether the major alterations detected in EM roots were promoted by the fungal partner. EM plants exhibited a more pronounced ability to maintain K+/Na+ homeostasis under salt stress. The influx of Na+ was reduced after short-term (50 mm NaCl, 24 h) and long-term (50 mm NaCl, 7 d) exposure to salt stress in mycorrhizal roots, especially in NAU associations. Flux data for P. involutus and susceptibility to Na+-transport inhibitors indicated that fungal colonization contributed to active Na+ extrusion and H+ uptake in the salinized roots of P. × canescens. Moreover, EM plants retained the ability to reduce the salt-induced K+ efflux, especially under long-term salinity. Our study suggests that P. involutus assists in maintaining K+ homeostasis by delivering this nutrient to host plants and slowing the loss of K+ under salt stress. EM P. × canescens plants exhibited an enhanced Ca2+ uptake ability, whereas short-term and long-term treatments caused a marked Ca2+ efflux from mycorrhizal roots, especially from NAU-colonized roots. We suggest that the release of additional Ca2+ mediated K+/Na+ homeostasis in EM plants under salt stress.

Exogenous hydrogen peroxide, nitric oxide and calcium mediate root ion fluxes in two non-secretor mangrove species subjected to NaCl stress

Using 3-month-old seedlings of Bruguiera gymnorrhiza (L.) Savigny and Kandelia candel (L.) Druce, we compared species differences in ionic homeostasis control between the two non-secretor mangrove species. A high salinity (400 mM NaCl, 4 weeks) resulted in a decline of the K(+)/Na(+) ratio in root and leaf tissues, and the reduction was more pronounced in K. candel (41-66%) as compared with B. gymnorrhiza (5-36%). Salt-altered flux profiles of Na(+), K(+), H(+) and Ca(2+) in roots and effects of exogenous hydrogen peroxide (H(2)O(2)), nitric oxide (NO) and Ca(2+) on root ion fluxes were examined in seedlings that were hydroponically treated short term with 100 mM NaCl (ST, 24 h) and long term with 200 mM NaCl (LT, 7 days). Short term and LT salinity resulted in Na(+) efflux and a correspondingly increased H(+) influx in roots of both species, although a more pronounced effect was observed in B. gymnorrhiza. The salt-enhanced exchange of Na(+) with H(+) was obviously inhibited by amiloride (a Na(+)/H(+) antiporter inhibitor) or sodium orthovanadate (a plasma membrane H(+)-ATPase inhibitor), indicating that the Na(+) efflux resulted from active Na(+) exclusion across the plasma membrane. Short term and LT salinity accelerated K(+) efflux in the two species, but K. candel exhibited a higher flux rate. The salt-induced K(+) efflux was markedly restricted by the K(+) channel blocker, tetraethylammonium chloride, indicating that the K(+) efflux is mediated by depolarization-activated channels, e.g., KORCs (outward rectifying K(+) channels) and NSCCs (non-selective cation channels). Exogenous H(2)O(2) application (10 mM) markedly increased the apparent Na(+) efflux and limited K(+) efflux in ST-treated roots, although H(2)O(2) caused a higher Na(+) efflux in B. gymnorrhiza roots. CaCl(2) (10 mM) reduced the efflux of K(+) in salinized roots of the two mangroves, but its enhancement of Na(+) efflux was found only in B. gymnorrhiza. Under ST treatment, sodium nitroprusside (SNP) (100 ∝M, an NO donor) increased Na(+) efflux at the root apex of the two species; however, its inhibition of K(+) loss was seen only in K. candel. Of note, NaCl caused an obvious influx of Ca(2+) in B. gymnorrhiza roots, which was enhanced by H(2)O(2) (10 mM). Therefore, the salt-induced Ca(2+) benefits B. gymnorrhiza in maintaining K(+)/Na(+) homeostasis under high external salinity.