Shurong Deng

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.

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.

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.

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.

Overexpression of copper/zinc superoxide dismutase from mangrove Kandelia candel in tobacco enhances salinity tolerance by the reduction of reactive oxygen species in chloroplast.

Na+ uptake and transport in Kandelia candel and antioxidative defense were investigated under rising NaCl stress from 100 to 300 mM. Salinized K. candel roots had a net Na+ efflux with a declined flux rate during an extended NaCl exposure. Na+ buildup in leaves enhanced H2O2 levels, superoxide dismutase (SOD) activity, and increased transcription of CSD gene encoding a Cu/Zn SOD. Sequence and subcellular localization analyses have revealed that KcCSD is a typical Cu/Zn SOD in chloroplast. The transgenic tobacco experimental system was used as a functional genetics model to test the effect of KcCSD on salinity tolerance. KcCSD-transgenic lines were more Na+ tolerant than wild-type (WT) tobacco in terms of lipid peroxidation, root growth, and survival rate. In the latter, 100 mM NaCl led to a remarkable reduction in chlorophyll content and a/b ratio, decreased maximal chlorophyll a fluorescence, and photochemical efficiency of photosystem II. NaCl stress in WT resulted from H2O2 burst in chloroplast. Na+ injury to chloroplast was less pronounced in KcCSD-transgenic plants due to upregulated antioxidant defense. KcCSD-transgenic tobacco enhanced SOD activity by an increment in SOD isoenzymes under 100 mM NaCl stress from 24 h to 7 day. Catalase activity rose in KcCSD overexpressing tobacco plants. KcCSD-transgenic plants better scavenged NaCl-elicited reactive oxygen species (ROS) compared to WT ones. In conclusion, K. candel effectively excluded Na+ in roots during a short exposure; and increased CSD expression to reduce ROS in chloroplast in a long-term and high saline environment.

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.

Overexpression of PeHA1 enhances hydrogen peroxide signaling in salt-stressed Arabidopsis

The plant plasma membrane (PM) H(+)-ATPase plays a crucial role in controlling K(+)/Na(+) homeostasis under salt stress. Our previous microarray analysis indicated that Populus euphratica retained a higher abundance of PM H(+)-ATPase transcript versus a salt-sensitive poplar. To clarify the roles of the PM H(+)-ATPase in salt sensing and adaptation, we isolated the PM H(+)-ATPase gene PeHA1 from P. euphratica and introduced it into Arabidopsis thaliana. Compared to wild-type, PeHA1-transgenic Arabidopsis had a greater germination rate, root length, and biomass under NaCl stress (50-150 mM). Ectopic expression of PeHA1 remarkably enhanced the capacity to control the homeostasis of ions and reactive oxygen species in salinized Arabidopsis. Flux data from salinized roots showed that transgenic plants exhibited a more pronounced Na(+)/H(+) antiport and less reduction of K(+) influx versus wild-type. Enhanced PM ATP hydrolytic activity, proton pumping, and Na(+)/H(+) antiport in PeHA1-transgenic plants, were consistent to those observed in vivo, i.e., H(+) extrusion, external acidification, and Na(+) efflux. Activities of the antioxidant enzymes ascorbate peroxidase and catalase were typically higher in transgenic seedlings irrespective of salt concentration. In transgenic Arabidopsis roots, H2O2 production was higher under control conditions and increased more rapidly than wild-type when plants were subjected to NaCl treatment. Interestingly, transgenic plants were unable to control K(+)/Na(+) homeostasis when salt-induced H2O2 production was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. These observations suggest that PeHA1 accelerates salt tolerance partially through rapid H2O2 production upon salt treatment, which triggers adjustments in K(+)/Na(+) homeostasis and antioxidant defense in Arabidopsis.

NaCl-elicited, vacuolar Ca2+ release facilitates prolonged cytosolic Ca2+ signaling in the salt response of Populus euphratica cells

High environmental salt elicits an increase in cytosolic Ca(2+) ([Ca(2+)]cyt) in plants, which is generated by extracellular Ca(2+) influx and Ca(2+) release from intracellular stores, such as vacuole and endoplasmic reticulum. This study aimed to determine the physiological mechanisms underlying Ca(2+) release from vacuoles and its role in ionic homeostasis in Populus euphratica. In vivo Ca(2+) imaging showed that NaCl treatment induced a rapid elevation in [Ca(2+)]cyt, which was accompanied by a subsequent release of vacuolar Ca(2+). In cell cultures, NaCl-altered intracellular Ca(2+) mobilization was abolished by antagonists of inositol (1, 4, 5) trisphosphate (IP3) and cyclic adenosine diphosphate ribose (cADPR) signaling pathways, but not by slow vacuolar (SV) channel blockers. Furthermore, the NaCl-induced vacuolar Ca(2+) release was dependent on extracellular ATP, extracellular Ca(2+) influx, H2O2, and NO. In vitro Ca(2+) flux recordings confirmed that IP3, cADPR, and Ca(2+) induced substantial Ca(2+) efflux from intact vacuoles, but this vacuolar Ca(2+) flux did not directly respond to ATP, H2O2, or NO. Moreover, the IP3/cADPR-mediated vacuolar Ca(2+) release enhanced the expression of salt-responsive genes that regulated a wide range of cellular processes required for ion homeostasis, including cytosolic K(+) maintenance, Na(+) and Cl(-) exclusion across the plasma membrane, and Na(+)/H(+) and Cl(-)/H(+) exchanges across the vacuolar membrane.

Extracellular ATP signaling and homeostasis in plant cells.

Extracellular ATP (eATP) is now recognized as an important signaling agent in plant growth and defense response to environmental stimuli. eATP has dual functions in plant cell signaling, which is largely dependent on its concentration in the extracellular matrix (ECM). A lethal level of eATP (extremely low or high) causes cell death, whereas a moderate level of eATP benefits plant growth and development. Ecto-apyrases (Nucleoside Triphosphate-Diphosphohydrolase) help control the eATP concentrations in the ECM, and thus contributing to the mediation of plant growth and defense response upon environmental stress. In this review, we summarize eATP signaling in plants and highlight the correlation between eATP homeostasis control and programmed cell death.