胡杨响应盐胁迫与离子平衡调控信号网络研究
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摘要
土壤盐渍化影响全世界的农林业生产,并危及生态环境,尤其是生态脆弱的干旱和半干旱地区。深入认识植物特别是典型植物耐盐生理和分子机制,对指导农林作物的抗逆性遗传改良具有重要意义。胡杨是我国西北干旱盐碱的荒漠和戈壁地带惟一能够形成森林的高大乔木树种,是进行木本植物抗逆性研究的典型材料。该树种具有很强的耐盐性,近年来很多国内外学者将胡杨作为耐盐的木本模式植物,从生理、生化和分子生物学等方面对其耐盐机理进行了较为系统的研究。
盐渍环境下植物能否维持细胞内的K+/Na+平衡对其适应高盐生境至关重要,而K+/Na+平衡是由一系列复杂的信号网络调控的。胁迫信使如钙(Ca2+)、过氧化氢(H202)和一氧化氮(NO)等都参与调控植物细胞的K+/Na+平衡。最近的研究证明,信号分子胞外ATP(eATP)参与植物的生物胁迫响应,但是,在盐胁迫下eATP是否参与调控植物的离子平衡仍然未知。目前,在生理水平上对胡杨K+/Na+平衡的研究仅仅局限在静态的离子积累和区隔化方面,有关K+/Na+平衡调控的信号网络仍不清楚。
因此,本论文重点研究胡杨响应盐胁迫的信号网络及离子平衡调控机理。在盐胁迫的适应机制方面,以耐盐的胡杨和不耐盐的群众杨水培苗为材料,利用非损伤微测技术(扫描离子选择性微电极技术,SIET)系统研究了NaCl胁迫下根组织和根细胞离子流(Na+、H+和K+)的动态变化,从动态离子转运的角度揭示了相关离子转运体和通道在胡杨根细胞K+/Na+平衡调控中的作用。在盐胁迫响应方面,(1)以胡杨和群众杨愈伤细胞为材料,利用能谱分析技术(EDAX)和激光共聚焦显微镜技术(Confocal)研究了盐诱导NO和H202在不同杨树细胞耐盐性中的作用；(2)以耐盐的胡杨愈伤细胞为模式系统,利用SIET、Confocal和EDAX等技术手段,并结合系统的药理学实验,探讨了胡杨细胞响应盐胁迫离子效应和渗透效应的信号网络：在离子胁迫响应方面,解析了PM H+-偶联转运体、H202和Ca2+信号在K+/Na+平衡调控中的作用,建立了胡杨细胞响应离子胁迫及K+/Na+平衡调控的信号途径；在渗透胁迫响应方面,发现eATP也参与调控胡杨细胞在盐胁迫下的K+/Na+平衡及抗氧化防御,同时发现胞外高浓度ATP能够诱导胡杨细胞主动程序化死亡(PCD),并提出了eATP诱导PCD的信号途径。本论文建立了胡杨响应NaCl胁迫及防御反应调控的信号网络。主要研究结果如下：
1.EDAX结果显示,在长期盐胁迫(50 mmol/L NaCl,3周)下胡杨根细胞比群众杨能够更有效地维持K+水平,并且能够限制Na+的积累。动态离子流的结果进一步证明了胡杨根细胞的K+/Na+平衡能力。SIET数据显示,胡杨根在瞬时和长期盐处理下K+外流都较群众杨弱。利用K+通道抑制剂TEA处理两种杨树根系,发现瞬时盐诱导的K+外流受到抑制,而质膜H+-ATPase抑制剂钒酸钠(Vanadate)却促进了盐诱导的K+外流,这表明杨树在盐胁迫下的根系K+外流是由去极化激活的外向K+通道(DA-KORCs)和非选择性阳离子通道(NSCCs)介导的。胡杨根系在长期盐胁迫下外排Na+能力强于群众杨,这源于其较强的质膜Na+/H+逆向运输活性。与群众杨相比,胡杨根尖能够迅速响应盐胁迫,H+外流迅速增强,表明胡杨根尖细胞PM质子泵被NaCl激活：质子泵一方面提供质子动力势来驱动质膜Na+/H+逆向运输;另一方面,降低NaCl诱导的质膜去极化程度,在减少Na+经由NSCCs内流的同时阻止了K+通过去极化激活K+通道的流失。此外,还从K+/Na+离子平衡调控的角度阐明了钙离子提高树木耐盐性的机理。两种杨树在外源Ca2+(10 mmol/L)处理下K+/Na+平衡能力都有所提高,而Ca2+对盐敏感杨树的效果更加明显。实验结果说明,Ca2+离子上调了根细胞质膜的质子泵活性：这不仅能驱动跨膜的Na+/H+逆向转运,同时还能够降低盐处理细胞质膜的去极化程度,限制了经由DA-KORCs和DA-NSCCs的外向K+流,有利于维持胞内的K+/Na+平衡。
2.在根细胞Na+外排方面,实验结果显示,无论是在短期(50 mmol/L NaCl,24h)还是长期盐胁迫(100 mmol/L NaCl,15天)下,胡杨根尖(0-3000μm)Na+外流和H+内流都显著高于盐敏感的群众杨。从长期盐处理的胡杨和群众杨根系游离出的原生质体也呈现同样的现象,而且,盐处理胡杨根原生质体Na+和H+的逆向流动在酸性环境下(pH 5.5)最为显著。利用质膜H+-ATPase抑制剂(钒酸钠)和Na+/H+逆向转运蛋白抑制剂阿米洛利(Amiloride)处理胡杨根尖和原生质体,发现二者均能同时抑制NaCl诱导的Na+外流和H+内流。这些结果证明了盐胁迫下胡杨根细胞的Na+外排是源于其质膜的Na+/H+逆向运输。与之比较,盐处理群众杨根尖和原生质体细胞的Na+/H+逆向运输能力极弱。实验结果还显示,NaCl所诱导的胡杨根细胞的离子转运具有离子特异性,且有别于渗透效应诱导的离子流。
3.比较研究了不同耐盐性杨树细胞对盐胁迫初始响应的差异。从胡杨和群众杨茎尖幼嫩组织诱导出愈伤细胞,对其进行NaCl处理,根据细胞活力、电解质外渗率和K+/Na+积累等生理指标判断,胡杨细胞的耐盐性显著强于群众杨,进而又探讨了NO和H202在NaCl胁迫下的产生模式及其与细胞耐盐性的关系。NaCl(150mmol/L)处理能迅速且大幅度地提高胡杨细胞H202和NO的水平,相反,群众杨细胞在初始盐胁迫下则无此响应,只是在胁迫后期H202水平才有所增加。胡杨细胞的抗氧化酶活性(抗坏血酸过氧化物酶/APX,超氧化物岐化酶/SOD,过氧化氢酶/CAT,谷胱甘肽还原酶/GR)在盐胁迫下显著提高,而群众杨细胞抗氧化酶活性却显著下降。在药理学实验中,当盐诱导的H202和NO受到抑制时,胡杨细胞的K+/Na+平衡调控能力和抗氧化酶活性也显著降低。实验结果证明盐诱导产生的NO和H202对胡杨细胞的K+/Na+平衡和抗氧化防御具有正向调控作用。
4.论文探讨了胡杨细胞对离子胁迫的响应机制。采用耐盐的胡杨愈伤细胞,研究了NaCl诱导的H2O2和Ca2+信号在K+/Na+平衡调控中的作用。SIET结果显示,NaCl处理后胡杨细胞呈现出较强的Na+/H+逆向转运活性,同时,盐胁迫也造成细胞质膜去极化和K+外流。当盐诱导产生的H202被抑制时,胡杨细胞的N+/H+逆向转运速率下降而K+的外流却明显增强。对于第二信使Ca2+,NaCl能够迅速诱导胞外Ca2+内流并使细胞质内自由Ca2+浓度增加。药理学实验证明,NaCl诱导的Ca2+信号参与了H202对胡杨细胞K+/Na+平衡的调控。NaCl、Cl-(choline Cl)、Na+(Na2SO4)等离子能够诱发H+的迅速内流,我们推断H+内流对胁迫信使的产生具有重要作用,这是由于钒酸钠(质膜H+-ATPase的抑制剂)和阿米洛利(Amiloride)(Na+/H+逆向转运蛋白的抑制剂)在抑制盐诱导H+内流的同时也限制了H202和Ca2+信号的产生,并且,盐处理胡杨细胞的K+/Na+平衡也不能维持。根据上述结果,我们推测胡杨细胞质膜H+偶联的转运蛋白能够响应离子胁迫而造成H+内流,H+内流导致了pH变化,从而激发了下游H202和Ca2+信号的产生,质膜质子泵和Na+/H+逆向转运蛋白随之被进一步激活,细胞内K+/Na+平衡得以维持。
5.研究了胡杨细胞对盐胁迫渗透效应的响应机制,建立了胡杨的eATP信号途径。eATP在动物系统中被证明是一种作用广泛的生理调节信号分子。在植物系统中,eATP已被证实能够调控植物生长、发育和生物胁迫响应,但对其在植物细胞盐胁迫响应过程中的作用属于未知。我们对胡杨细胞进行渗透胁迫处理,发现NaCl和等渗甘露醇处理都能够提高细胞外基质中的ATP浓度([eATP]),但[eATP]在20 min后即恢复至对照水平。己糖激酶和葡萄糖系统(H-G system)能够消耗胞外的ATP,因而阻止了NaCl和甘露醇诱导的eATP水平的增加。药理学的实验结果表明,eATP在调控胡杨细胞耐盐性方面具有重要作用：(1)动物细胞质膜P2受体的拮抗剂(suramin)和H-G system显著降低了胡杨细胞在NaCl胁迫(200 mmol/L 24h)和渗透胁迫(340mmol/L甘露醇24h)下的细胞活力,并显著增强了H202的积累；(2)在盐胁迫下,suramin和H-G system处理还明显提高了Na+在胡杨细胞质中的浓度,同时降低了Na+在液泡中的积累；(3)抑制剂处理的胡杨细胞在盐处理后,质膜去极化程度加剧,且K+外流幅度也有所增强。实验还发现,在渗透胁迫(NaCl和甘露醇)下,P2受体的拮抗剂和H-G system虽然明显限制了H202和Ca2+信使的产生,却没有改变H+的跨膜运输。所以,我们的实验结果表明,细胞内ATP向胞外释放是由NaCl的渗透效应导致,而非离子效应,eATP经质膜P2受体感知后,激发下游H202和Ca2+信号的产生,从而进一步调控K+、Na+转运体和抗氧化防御,使盐处理细胞的K+/Na+平衡和活性氧平衡得以维持。
6.研究了eATP诱导胡杨细胞程序化死亡(PCD)的生理机制。作为信号信使,eATP在胡杨响应初始盐胁迫过程中至关重要。但我们发现,NaCl诱导的eATP水平升高仅仅维持了20min。在动物中,高水平的eATP能够诱发PCD。因此,我们认为持续高水平的eATP可能会对胡杨细胞的生理状态产生负面影响。我们的研究发现,高浓度eATP(0.5-2.0 mmol/L)促进了胡杨细胞的凋亡,且凋亡率依赖于ATP处理的浓度和时间。特别是发现凋亡细胞呈现出PCD的标志性事件,如细胞质收缩、染色质浓缩、DNA片段化等。通过药理学实验,我们发现eATP诱导的PCD主要包括了一系列信号事件：如质膜嘌呤受体的激活、胞外Ca2+内流、液泡Ca2+释放、线粒体Ca2+吸收、线粒体H202爆发、线粒体超级化等,而诱发PCD的重要因素,如细胞色素c释放、内源ATP含量的增加和类凋亡酶活性的提高都依赖于上述信号事件。值得注意的是,eATP处理明显增加了内源ATP的含量和类凋亡酶活性,还促进了不依赖于mPTP(线粒体膜转换孔)的细胞色素c释放。研究还发现,eATP处理能够显著增强细胞内源的NO水平,但NO为冗余信号,并不参与eATP-PCD过程。综上,我们提出了eATP诱导胡杨细胞发生PCD的信号途径。
总之,实验结果进一步证实,胡杨细胞和组织具有很强的K+/Na+平衡调控能力。其中,Na+平衡的维持在很大程度上源自排Na+能力,这种排盐能力依赖于胡杨质膜较强的质子泵和Na+/H+逆向运输活性。胡杨根细胞的这种排盐能力使Na+在根中的径向运输和根冠纵向运输下降。胡杨细胞K+平衡的维持也与其较强的质膜质子泵活性有关,胡杨质膜H+-ATPase能在盐胁迫下降低质膜的去极化程度,减少了K+通过去极化激活K+通道的流失。在细胞对盐胁迫的响应上,发现eATP和质膜H+偶联转运体分别感知渗透胁迫效应和离子胁迫效应,通过各自的信号转导途径激活下游H2O2和Ca2+信号, H2O2和Ca2+进一步调控质膜或液泡膜K+、Na+相关转运体或通道来维持合适的K+/Na+平衡。在长期胁迫下,由于胞外三磷酸核苷酸水解酶(Apyrase)的水解作用,胡杨能维持eATP在合理水平,从而避免了PCD的发生,使胡杨细胞能适应长期盐胁迫环境。
Soil salinity causes increasingly agricultural and environmental problems on a worldwide scale, especially in arid areas. Understanding physiological and molecular mechanisms of salt tolerance is important for the improvement of plant productivity under salinity conditions. Populus euphratica Oliv. (P. euphratica) is a valuable tree species used for afforestation on saline and alkaline desert sits, and plays very important roles in stabilizing sand dunes, and in agriculture shelter belt construction in north-west China. P. euphratica has a higher capacity to tolerate salinity, and in recent years it has been widely considered as a model woody plant to address tree-specific questions—especially physiological, biochemical and molecular mechanisms in salt tolerance.
Maintenance of intracellular K+/Na+ homeostasis is crucial for plants to adapt to saline environments. The regulation of K+/Na+ homeostasis is complicated by a complex signaling network. For instance, numerous signaling molecules, such as Ca2+, hydrogen peroxide (H2O2) and nitric oxide (NO), play a regulating role in K+/Na+ homeostasis in model plants. Recently, a novel signaling molecule, extracellular ATP (eATP), has been reported to be involved in the responses of plant cells to biotic stress. However, the roles of eATP in mediating plant salt tolerance and K+/Na+ homeostasis are largely unknown. At present, many investigations related to steady ion relations always focus on salt accumulation and cellular ion compartmentation, however, the signaling network in the mediation of K+/Na+homeostasis in P. euphratica is still unclear.
In this study, we attempt to elucidate the signaling network in the perception of salt stress and K+/Na+ homeostasis regulation in P. euphratica. Using hydroponic seedlings of salt-resistant P. euphratica and salt-sensitive P. popularis 35-44 (P. popularis), we investigated the NaCl-induced alterations of cellular and tissue ion fluxes in roots (Na+, H+ and K+) by means of scanning ion-selective micro-electrode technique (SIET). We explored the contributions of plasma membrane (PM) H+-coupled transporters and channels to the K+/Na+ homeostasis mediation in P. euphratica. Callus cells were initiated from P. euphratica and P. popularis shoots and used to address the perception of poplar to salt stress. The contributions of H2O2 and NO to K+/Na+ homeostasis and antioxidant defense were elucidated in the two contrasting poplars by means of EDAX (energy dispersive X-ray analysis) and Confocal laser scanning microscopy. Using EDAX, confocal and SIET, we designed a variety of pharmacological experiments to clarify the differential response of P. euphratica to osmotic and ion-specific effects of NaCl. We confirmed the involvement of PM H+-coupled transporters, H2O2 and Ca2+ in the mediation of K+/Na+ homeostasis in P. euphratica cells, and a cellular signaling model upon ion specific effect was proposed. The role of salt-induced eATP (extracellular ATP) signaling in K+/Na+ homeostasis control and antioxidant defence were also explored in NaCl-stressed P. euphratica cells. Finally, we found that an excess eATP induced PCD (programmed cell death) in P. euphratica cells and a signaling pathway of eATP-PCD was proposed. Taken together, we proposed a signaling network to elucidate the perception and defense when P. euphratica cells were subjected to NaCl salinity.
The main experimental results and conclusions are as follows:
1. Compared to P. popularis, P. euphratica roots exhibited a greater capacity to retain K+ and to restrict Na+ accumulation after exposure to a long-term (LT) salinity (50 mM NaCl,3 weeks) by means of EDAX and ion-flux measurements. Our SIET data show that P. euphratica roots retained a lesser K+ efflux under both a short-and long-term of salt stress, as compared to P. popularis. Salt shock (SS)-induced K+ efflux in the two species was markedly restricted by K+ channels blocker, TEA (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 channels, e.g. KORCs (outwarding rectifying K+ channels), and NSCCs (non-selective cation channels). P. euphratica roots were more effectively to exclude Na+ than P. popularis in a 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 salinised 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. Exogenously applied Ca2+ was favorable for poplar roots to maintain K+/Na+ homeostasis and the effect was more pronounced in the salt-sensitive species. Ca2+ application markedly limited salt-induced K+ efflux but enhanced the apparent Na+ efflux, thus enables the two species, especially the salt-sensitive poplar, to retain K+/Na+ homeostasis in roots exposed to prolonged NaCl treatment.
2. Compared to P. popularis, P. euphratica roots (0-3000μm from the apex) exhibited a higher capacity to extrude Na+ after a short-term exposure to 50mM NaCl (24h) and a long term in a saline environment of 100mM 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. The pattern of NaCl-induced fluxes of H+ and Na+ 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.
3. We found the species difference in the cellular response to NaCl treatment. Using callus cells of a salt-tolerant P. euphratica and a salt-sensitive P. popularis, the effects of NaCl stress on hydrogen peroxide (H2O2) 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 (150mM) caused a rapid increase of H2O2 and NO in P. euphratica cells. Moreover, salinised P. euphratica cells retained a high and stable level of H2O2 and NO during the period of 24-h salt stress. However, there were no evident increase of H2O2 and NO in P. popularis after the onset of salinity and an increase of H2O2 was only seen after a prolonged period of salt treatment. 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 H2O2 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 H2O2 and NO enable P. euphratica cells to regulate ionic and ROS (reactive oxygen species) homeostasis under salinity stress in the longer term.
4. With regard to the cellular response to ion-specific effects, 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 P. euphratica. SIET data showed an obvious Na+/H+ antiport in salinized cells; Meanwhile, NaCl stress caused a net K+ efflux, because of the salt-induced membrane depolarization. H2O2 levels, upwards regulated by salinity, contributed to ionic homeostasis, because H2O2 restrictions by DPI or DMTU caused an enhanced K+ efflux and decreased Na+/H+ antiport activity. NaCl induced a net Ca2+ influx and a subsequent rise of free Ca2+ in the cytosol ([Ca2+]cyt), which is involved in H2O2-mediated K+/Na+ homeostasis in salinized P. euphratica cells. NaCl, Cl- (choline Cl) and Na+(Na2SO4) caused a net H+ influx, which was presumably able to trigger the production of stress signals. When callus cells were pretreated with inhibitors of the Na+/H+ antiport system, the NaCl-induced elevation of H2O2 and [Ca+]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 mediated H+ translocation upon salt treatment and brought about an alternation of pH. The pH variation 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 signalling pathway in the response of P. euphratica cells to ion-specific effects is proposed.
5. We investigated the cellular response to osmotic effects and an extracellular ATP (eATP) signaling pathway was established in P. euphratica. It is well known that eATP plays a versatile signaling role in animals, and now emerging evidence shows that it regulates higher plant growth, development and biotic responses. Whether eATP is involved in plant salinity sensing and adaptation is still unknown. Thus, using callus cells of P. euphratica, we attempt to clarify this issue in the present study. NaCl (200 mM) and iso-osmotic mannitol induced a rapid increase of ATP level in extracellular medium within 20 minutes. Hexokinase and glucose system (H-G system) could hydrolysis ATP rapidly and thus blocked the salt-and mannitol-induced elevation of eATP. Pharmacological studies show that eATP plays a regulating role the salt resistance of P. euphratica. Application of antagonists of animal PM P2 receptors and H-G system significantly decreased the cell viability in stressed P. euphratica cells, but enhanced H2O2 accumulation after exposure of 24 h. Under salt stress conditions, the treatment of suramin (P2 receptor antagonist) and H-G system increased Na+ accumulation in the cytoplasma but decreased Na+ compartmentation in the vacuole. Meanwhile, the inhibitors of eATP (suramin, H-G system) enhanced K+ efflux and PM depolarization in salt-stressed P. euphratica cells. After the application of antagonist of animal PM P2 receptors and H-G system, the early responses of H2O2 and Ca2+ induced by NaCl and mannitol were impaired, whereas there were no corresponding changes in H+ fluxes. Therefore, our results suggests that NaCl (osmotic effect) induced a release of endogenous ATP, which is perceive by purinoceptors in the PM, leading to the induction of downstream signals, e.g. H2O2 and cytosolic Ca2+, that are required for the regulation of K+ and Na+ transporters and antioxidant defence. Consequently, K+/Na+ and ROS homeostasis of P. euphratica were maintained during a prolonged period of salt stress.
6. The physiological mechanism of PCD that induced by eATP was explored.. It has shown that eATP plays a crucial role in mediating the salinity tolerance of P. euphratica cells. However, the NaCl-induced eATP was a transient response and eATP returned to pretreatment levels after 20 min of salt and mannitol treatment. We suppose that eATP may exert adverse effects on the woody species since an excessive eATP induces PCD in animal cells. In our study, exogenously applied ATP (high dose,0.5 to 2 mM) resulted in a dose- and time-dependent reduction of viability and the agonist-treated cells displayed hallmark features indicative of PCD, such as cytoplasmic shrinkage, chromatin condensation and DNA fragmentation. A sequence of events accounting for ATP-induced PCD is proposed as evidenced using a variety of pharmacological agents. Extracellular ATP (eATP) caused an elevation of Ca2+ in the cytosol ([Ca2+]cyt), resulting from a transient influx of Ca2+ across the plasma membrane (PM) and a subsequent release of Ca2+ from the vacuole. The long-term sustained [Ca2+]cyt resulted in an evident Ca2+ uptake in the mitochondria, leading to a H2O2 accumulation therein. Noteworthy is that P. euphratica exhibited an increased mitochondrial transmembrane potential (△Ψm) and the release of cytochrome c took place without the opening of permeability transition pore over the period of ATP stimulation. Moreover, the eATP-induced increase of intracellular ATP, which is essential for the activation of caspase-like proteases and the subsequent PCD execution, was found to be correlated with the increased△Ψm. NO is implicated as a downstream component of [Ca2+]cyt but plays a negligible role in eATP-stimulated cell death. We speculate that ATP is assumed to bind P2-like receptors in the PM, leading to the induction of downstream intermediate signals because the proposed sequence of events in PCD signaling chain were terminated by an animal P2 receptor antagonist suramin.
In conclusion, at tissue and cellular levels, our data confirmed that the salt tolerance of P. euphratica is partly due to its strong ability on K+/Na+ homeostasis control. The Na+ extrusion is mainly ascribed to the strong activity of PM Na+/H+ antiport system (PM H+-ATPase and Na+/H+ antiporter) and thus, P. euphratica could restrict the radial transport of Na+ and decrease the accumulation of Na+ in the shoots and leaves. The maintenance of K+ homeostasis is mainly due to the higher activity of PM H+-ATPase, which decreased the magnitude of PM depolarization and consequently reduced the K+ loss through depolarization-activated K+ channels. At cellular level, the putative PM ATP receptors and H+-coupled ion transporters could sense osmotic and ionic effects of NaCl, respectively, and then independently activates H2O2 and Ca2+ signaling pathways. H2O2 and Ca2+ contribute to the up-regulation of K+- and Na+-related antiport system and ion channels in the PM and tonoplast, leading to a K+/Na+ homeostasis in salinised cells. In P. euphratica cells, the NaCl-induced release of ATP was hydrolysed by ecto-apyrase and the occurrence of eATP-induced PCD was avoided during a prolonged salt treatment. Finally, P. euphratica cells could survive the saline conditions under a long-term of salt stress.
盐渍环境下植物能否维持细胞内的K+/Na+平衡对其适应高盐生境至关重要,而K+/Na+平衡是由一系列复杂的信号网络调控的。胁迫信使如钙(Ca2+)、过氧化氢(H202)和一氧化氮(NO)等都参与调控植物细胞的K+/Na+平衡。最近的研究证明,信号分子胞外ATP(eATP)参与植物的生物胁迫响应,但是,在盐胁迫下eATP是否参与调控植物的离子平衡仍然未知。目前,在生理水平上对胡杨K+/Na+平衡的研究仅仅局限在静态的离子积累和区隔化方面,有关K+/Na+平衡调控的信号网络仍不清楚。
因此,本论文重点研究胡杨响应盐胁迫的信号网络及离子平衡调控机理。在盐胁迫的适应机制方面,以耐盐的胡杨和不耐盐的群众杨水培苗为材料,利用非损伤微测技术(扫描离子选择性微电极技术,SIET)系统研究了NaCl胁迫下根组织和根细胞离子流(Na+、H+和K+)的动态变化,从动态离子转运的角度揭示了相关离子转运体和通道在胡杨根细胞K+/Na+平衡调控中的作用。在盐胁迫响应方面,(1)以胡杨和群众杨愈伤细胞为材料,利用能谱分析技术(EDAX)和激光共聚焦显微镜技术(Confocal)研究了盐诱导NO和H202在不同杨树细胞耐盐性中的作用；(2)以耐盐的胡杨愈伤细胞为模式系统,利用SIET、Confocal和EDAX等技术手段,并结合系统的药理学实验,探讨了胡杨细胞响应盐胁迫离子效应和渗透效应的信号网络：在离子胁迫响应方面,解析了PM H+-偶联转运体、H202和Ca2+信号在K+/Na+平衡调控中的作用,建立了胡杨细胞响应离子胁迫及K+/Na+平衡调控的信号途径；在渗透胁迫响应方面,发现eATP也参与调控胡杨细胞在盐胁迫下的K+/Na+平衡及抗氧化防御,同时发现胞外高浓度ATP能够诱导胡杨细胞主动程序化死亡(PCD),并提出了eATP诱导PCD的信号途径。本论文建立了胡杨响应NaCl胁迫及防御反应调控的信号网络。主要研究结果如下：
1.EDAX结果显示,在长期盐胁迫(50 mmol/L NaCl,3周)下胡杨根细胞比群众杨能够更有效地维持K+水平,并且能够限制Na+的积累。动态离子流的结果进一步证明了胡杨根细胞的K+/Na+平衡能力。SIET数据显示,胡杨根在瞬时和长期盐处理下K+外流都较群众杨弱。利用K+通道抑制剂TEA处理两种杨树根系,发现瞬时盐诱导的K+外流受到抑制,而质膜H+-ATPase抑制剂钒酸钠(Vanadate)却促进了盐诱导的K+外流,这表明杨树在盐胁迫下的根系K+外流是由去极化激活的外向K+通道(DA-KORCs)和非选择性阳离子通道(NSCCs)介导的。胡杨根系在长期盐胁迫下外排Na+能力强于群众杨,这源于其较强的质膜Na+/H+逆向运输活性。与群众杨相比,胡杨根尖能够迅速响应盐胁迫,H+外流迅速增强,表明胡杨根尖细胞PM质子泵被NaCl激活：质子泵一方面提供质子动力势来驱动质膜Na+/H+逆向运输;另一方面,降低NaCl诱导的质膜去极化程度,在减少Na+经由NSCCs内流的同时阻止了K+通过去极化激活K+通道的流失。此外,还从K+/Na+离子平衡调控的角度阐明了钙离子提高树木耐盐性的机理。两种杨树在外源Ca2+(10 mmol/L)处理下K+/Na+平衡能力都有所提高,而Ca2+对盐敏感杨树的效果更加明显。实验结果说明,Ca2+离子上调了根细胞质膜的质子泵活性：这不仅能驱动跨膜的Na+/H+逆向转运,同时还能够降低盐处理细胞质膜的去极化程度,限制了经由DA-KORCs和DA-NSCCs的外向K+流,有利于维持胞内的K+/Na+平衡。
2.在根细胞Na+外排方面,实验结果显示,无论是在短期(50 mmol/L NaCl,24h)还是长期盐胁迫(100 mmol/L NaCl,15天)下,胡杨根尖(0-3000μm)Na+外流和H+内流都显著高于盐敏感的群众杨。从长期盐处理的胡杨和群众杨根系游离出的原生质体也呈现同样的现象,而且,盐处理胡杨根原生质体Na+和H+的逆向流动在酸性环境下(pH 5.5)最为显著。利用质膜H+-ATPase抑制剂(钒酸钠)和Na+/H+逆向转运蛋白抑制剂阿米洛利(Amiloride)处理胡杨根尖和原生质体,发现二者均能同时抑制NaCl诱导的Na+外流和H+内流。这些结果证明了盐胁迫下胡杨根细胞的Na+外排是源于其质膜的Na+/H+逆向运输。与之比较,盐处理群众杨根尖和原生质体细胞的Na+/H+逆向运输能力极弱。实验结果还显示,NaCl所诱导的胡杨根细胞的离子转运具有离子特异性,且有别于渗透效应诱导的离子流。
3.比较研究了不同耐盐性杨树细胞对盐胁迫初始响应的差异。从胡杨和群众杨茎尖幼嫩组织诱导出愈伤细胞,对其进行NaCl处理,根据细胞活力、电解质外渗率和K+/Na+积累等生理指标判断,胡杨细胞的耐盐性显著强于群众杨,进而又探讨了NO和H202在NaCl胁迫下的产生模式及其与细胞耐盐性的关系。NaCl(150mmol/L)处理能迅速且大幅度地提高胡杨细胞H202和NO的水平,相反,群众杨细胞在初始盐胁迫下则无此响应,只是在胁迫后期H202水平才有所增加。胡杨细胞的抗氧化酶活性(抗坏血酸过氧化物酶/APX,超氧化物岐化酶/SOD,过氧化氢酶/CAT,谷胱甘肽还原酶/GR)在盐胁迫下显著提高,而群众杨细胞抗氧化酶活性却显著下降。在药理学实验中,当盐诱导的H202和NO受到抑制时,胡杨细胞的K+/Na+平衡调控能力和抗氧化酶活性也显著降低。实验结果证明盐诱导产生的NO和H202对胡杨细胞的K+/Na+平衡和抗氧化防御具有正向调控作用。
4.论文探讨了胡杨细胞对离子胁迫的响应机制。采用耐盐的胡杨愈伤细胞,研究了NaCl诱导的H2O2和Ca2+信号在K+/Na+平衡调控中的作用。SIET结果显示,NaCl处理后胡杨细胞呈现出较强的Na+/H+逆向转运活性,同时,盐胁迫也造成细胞质膜去极化和K+外流。当盐诱导产生的H202被抑制时,胡杨细胞的N+/H+逆向转运速率下降而K+的外流却明显增强。对于第二信使Ca2+,NaCl能够迅速诱导胞外Ca2+内流并使细胞质内自由Ca2+浓度增加。药理学实验证明,NaCl诱导的Ca2+信号参与了H202对胡杨细胞K+/Na+平衡的调控。NaCl、Cl-(choline Cl)、Na+(Na2SO4)等离子能够诱发H+的迅速内流,我们推断H+内流对胁迫信使的产生具有重要作用,这是由于钒酸钠(质膜H+-ATPase的抑制剂)和阿米洛利(Amiloride)(Na+/H+逆向转运蛋白的抑制剂)在抑制盐诱导H+内流的同时也限制了H202和Ca2+信号的产生,并且,盐处理胡杨细胞的K+/Na+平衡也不能维持。根据上述结果,我们推测胡杨细胞质膜H+偶联的转运蛋白能够响应离子胁迫而造成H+内流,H+内流导致了pH变化,从而激发了下游H202和Ca2+信号的产生,质膜质子泵和Na+/H+逆向转运蛋白随之被进一步激活,细胞内K+/Na+平衡得以维持。
5.研究了胡杨细胞对盐胁迫渗透效应的响应机制,建立了胡杨的eATP信号途径。eATP在动物系统中被证明是一种作用广泛的生理调节信号分子。在植物系统中,eATP已被证实能够调控植物生长、发育和生物胁迫响应,但对其在植物细胞盐胁迫响应过程中的作用属于未知。我们对胡杨细胞进行渗透胁迫处理,发现NaCl和等渗甘露醇处理都能够提高细胞外基质中的ATP浓度([eATP]),但[eATP]在20 min后即恢复至对照水平。己糖激酶和葡萄糖系统(H-G system)能够消耗胞外的ATP,因而阻止了NaCl和甘露醇诱导的eATP水平的增加。药理学的实验结果表明,eATP在调控胡杨细胞耐盐性方面具有重要作用：(1)动物细胞质膜P2受体的拮抗剂(suramin)和H-G system显著降低了胡杨细胞在NaCl胁迫(200 mmol/L 24h)和渗透胁迫(340mmol/L甘露醇24h)下的细胞活力,并显著增强了H202的积累；(2)在盐胁迫下,suramin和H-G system处理还明显提高了Na+在胡杨细胞质中的浓度,同时降低了Na+在液泡中的积累；(3)抑制剂处理的胡杨细胞在盐处理后,质膜去极化程度加剧,且K+外流幅度也有所增强。实验还发现,在渗透胁迫(NaCl和甘露醇)下,P2受体的拮抗剂和H-G system虽然明显限制了H202和Ca2+信使的产生,却没有改变H+的跨膜运输。所以,我们的实验结果表明,细胞内ATP向胞外释放是由NaCl的渗透效应导致,而非离子效应,eATP经质膜P2受体感知后,激发下游H202和Ca2+信号的产生,从而进一步调控K+、Na+转运体和抗氧化防御,使盐处理细胞的K+/Na+平衡和活性氧平衡得以维持。
6.研究了eATP诱导胡杨细胞程序化死亡(PCD)的生理机制。作为信号信使,eATP在胡杨响应初始盐胁迫过程中至关重要。但我们发现,NaCl诱导的eATP水平升高仅仅维持了20min。在动物中,高水平的eATP能够诱发PCD。因此,我们认为持续高水平的eATP可能会对胡杨细胞的生理状态产生负面影响。我们的研究发现,高浓度eATP(0.5-2.0 mmol/L)促进了胡杨细胞的凋亡,且凋亡率依赖于ATP处理的浓度和时间。特别是发现凋亡细胞呈现出PCD的标志性事件,如细胞质收缩、染色质浓缩、DNA片段化等。通过药理学实验,我们发现eATP诱导的PCD主要包括了一系列信号事件：如质膜嘌呤受体的激活、胞外Ca2+内流、液泡Ca2+释放、线粒体Ca2+吸收、线粒体H202爆发、线粒体超级化等,而诱发PCD的重要因素,如细胞色素c释放、内源ATP含量的增加和类凋亡酶活性的提高都依赖于上述信号事件。值得注意的是,eATP处理明显增加了内源ATP的含量和类凋亡酶活性,还促进了不依赖于mPTP(线粒体膜转换孔)的细胞色素c释放。研究还发现,eATP处理能够显著增强细胞内源的NO水平,但NO为冗余信号,并不参与eATP-PCD过程。综上,我们提出了eATP诱导胡杨细胞发生PCD的信号途径。
总之,实验结果进一步证实,胡杨细胞和组织具有很强的K+/Na+平衡调控能力。其中,Na+平衡的维持在很大程度上源自排Na+能力,这种排盐能力依赖于胡杨质膜较强的质子泵和Na+/H+逆向运输活性。胡杨根细胞的这种排盐能力使Na+在根中的径向运输和根冠纵向运输下降。胡杨细胞K+平衡的维持也与其较强的质膜质子泵活性有关,胡杨质膜H+-ATPase能在盐胁迫下降低质膜的去极化程度,减少了K+通过去极化激活K+通道的流失。在细胞对盐胁迫的响应上,发现eATP和质膜H+偶联转运体分别感知渗透胁迫效应和离子胁迫效应,通过各自的信号转导途径激活下游H2O2和Ca2+信号, H2O2和Ca2+进一步调控质膜或液泡膜K+、Na+相关转运体或通道来维持合适的K+/Na+平衡。在长期胁迫下,由于胞外三磷酸核苷酸水解酶(Apyrase)的水解作用,胡杨能维持eATP在合理水平,从而避免了PCD的发生,使胡杨细胞能适应长期盐胁迫环境。
Soil salinity causes increasingly agricultural and environmental problems on a worldwide scale, especially in arid areas. Understanding physiological and molecular mechanisms of salt tolerance is important for the improvement of plant productivity under salinity conditions. Populus euphratica Oliv. (P. euphratica) is a valuable tree species used for afforestation on saline and alkaline desert sits, and plays very important roles in stabilizing sand dunes, and in agriculture shelter belt construction in north-west China. P. euphratica has a higher capacity to tolerate salinity, and in recent years it has been widely considered as a model woody plant to address tree-specific questions—especially physiological, biochemical and molecular mechanisms in salt tolerance.
Maintenance of intracellular K+/Na+ homeostasis is crucial for plants to adapt to saline environments. The regulation of K+/Na+ homeostasis is complicated by a complex signaling network. For instance, numerous signaling molecules, such as Ca2+, hydrogen peroxide (H2O2) and nitric oxide (NO), play a regulating role in K+/Na+ homeostasis in model plants. Recently, a novel signaling molecule, extracellular ATP (eATP), has been reported to be involved in the responses of plant cells to biotic stress. However, the roles of eATP in mediating plant salt tolerance and K+/Na+ homeostasis are largely unknown. At present, many investigations related to steady ion relations always focus on salt accumulation and cellular ion compartmentation, however, the signaling network in the mediation of K+/Na+homeostasis in P. euphratica is still unclear.
In this study, we attempt to elucidate the signaling network in the perception of salt stress and K+/Na+ homeostasis regulation in P. euphratica. Using hydroponic seedlings of salt-resistant P. euphratica and salt-sensitive P. popularis 35-44 (P. popularis), we investigated the NaCl-induced alterations of cellular and tissue ion fluxes in roots (Na+, H+ and K+) by means of scanning ion-selective micro-electrode technique (SIET). We explored the contributions of plasma membrane (PM) H+-coupled transporters and channels to the K+/Na+ homeostasis mediation in P. euphratica. Callus cells were initiated from P. euphratica and P. popularis shoots and used to address the perception of poplar to salt stress. The contributions of H2O2 and NO to K+/Na+ homeostasis and antioxidant defense were elucidated in the two contrasting poplars by means of EDAX (energy dispersive X-ray analysis) and Confocal laser scanning microscopy. Using EDAX, confocal and SIET, we designed a variety of pharmacological experiments to clarify the differential response of P. euphratica to osmotic and ion-specific effects of NaCl. We confirmed the involvement of PM H+-coupled transporters, H2O2 and Ca2+ in the mediation of K+/Na+ homeostasis in P. euphratica cells, and a cellular signaling model upon ion specific effect was proposed. The role of salt-induced eATP (extracellular ATP) signaling in K+/Na+ homeostasis control and antioxidant defence were also explored in NaCl-stressed P. euphratica cells. Finally, we found that an excess eATP induced PCD (programmed cell death) in P. euphratica cells and a signaling pathway of eATP-PCD was proposed. Taken together, we proposed a signaling network to elucidate the perception and defense when P. euphratica cells were subjected to NaCl salinity.
The main experimental results and conclusions are as follows:
1. Compared to P. popularis, P. euphratica roots exhibited a greater capacity to retain K+ and to restrict Na+ accumulation after exposure to a long-term (LT) salinity (50 mM NaCl,3 weeks) by means of EDAX and ion-flux measurements. Our SIET data show that P. euphratica roots retained a lesser K+ efflux under both a short-and long-term of salt stress, as compared to P. popularis. Salt shock (SS)-induced K+ efflux in the two species was markedly restricted by K+ channels blocker, TEA (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 channels, e.g. KORCs (outwarding rectifying K+ channels), and NSCCs (non-selective cation channels). P. euphratica roots were more effectively to exclude Na+ than P. popularis in a 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 salinised 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. Exogenously applied Ca2+ was favorable for poplar roots to maintain K+/Na+ homeostasis and the effect was more pronounced in the salt-sensitive species. Ca2+ application markedly limited salt-induced K+ efflux but enhanced the apparent Na+ efflux, thus enables the two species, especially the salt-sensitive poplar, to retain K+/Na+ homeostasis in roots exposed to prolonged NaCl treatment.
2. Compared to P. popularis, P. euphratica roots (0-3000μm from the apex) exhibited a higher capacity to extrude Na+ after a short-term exposure to 50mM NaCl (24h) and a long term in a saline environment of 100mM 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. The pattern of NaCl-induced fluxes of H+ and Na+ 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.
3. We found the species difference in the cellular response to NaCl treatment. Using callus cells of a salt-tolerant P. euphratica and a salt-sensitive P. popularis, the effects of NaCl stress on hydrogen peroxide (H2O2) 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 (150mM) caused a rapid increase of H2O2 and NO in P. euphratica cells. Moreover, salinised P. euphratica cells retained a high and stable level of H2O2 and NO during the period of 24-h salt stress. However, there were no evident increase of H2O2 and NO in P. popularis after the onset of salinity and an increase of H2O2 was only seen after a prolonged period of salt treatment. 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 H2O2 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 H2O2 and NO enable P. euphratica cells to regulate ionic and ROS (reactive oxygen species) homeostasis under salinity stress in the longer term.
4. With regard to the cellular response to ion-specific effects, 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 P. euphratica. SIET data showed an obvious Na+/H+ antiport in salinized cells; Meanwhile, NaCl stress caused a net K+ efflux, because of the salt-induced membrane depolarization. H2O2 levels, upwards regulated by salinity, contributed to ionic homeostasis, because H2O2 restrictions by DPI or DMTU caused an enhanced K+ efflux and decreased Na+/H+ antiport activity. NaCl induced a net Ca2+ influx and a subsequent rise of free Ca2+ in the cytosol ([Ca2+]cyt), which is involved in H2O2-mediated K+/Na+ homeostasis in salinized P. euphratica cells. NaCl, Cl- (choline Cl) and Na+(Na2SO4) caused a net H+ influx, which was presumably able to trigger the production of stress signals. When callus cells were pretreated with inhibitors of the Na+/H+ antiport system, the NaCl-induced elevation of H2O2 and [Ca+]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 mediated H+ translocation upon salt treatment and brought about an alternation of pH. The pH variation 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 signalling pathway in the response of P. euphratica cells to ion-specific effects is proposed.
5. We investigated the cellular response to osmotic effects and an extracellular ATP (eATP) signaling pathway was established in P. euphratica. It is well known that eATP plays a versatile signaling role in animals, and now emerging evidence shows that it regulates higher plant growth, development and biotic responses. Whether eATP is involved in plant salinity sensing and adaptation is still unknown. Thus, using callus cells of P. euphratica, we attempt to clarify this issue in the present study. NaCl (200 mM) and iso-osmotic mannitol induced a rapid increase of ATP level in extracellular medium within 20 minutes. Hexokinase and glucose system (H-G system) could hydrolysis ATP rapidly and thus blocked the salt-and mannitol-induced elevation of eATP. Pharmacological studies show that eATP plays a regulating role the salt resistance of P. euphratica. Application of antagonists of animal PM P2 receptors and H-G system significantly decreased the cell viability in stressed P. euphratica cells, but enhanced H2O2 accumulation after exposure of 24 h. Under salt stress conditions, the treatment of suramin (P2 receptor antagonist) and H-G system increased Na+ accumulation in the cytoplasma but decreased Na+ compartmentation in the vacuole. Meanwhile, the inhibitors of eATP (suramin, H-G system) enhanced K+ efflux and PM depolarization in salt-stressed P. euphratica cells. After the application of antagonist of animal PM P2 receptors and H-G system, the early responses of H2O2 and Ca2+ induced by NaCl and mannitol were impaired, whereas there were no corresponding changes in H+ fluxes. Therefore, our results suggests that NaCl (osmotic effect) induced a release of endogenous ATP, which is perceive by purinoceptors in the PM, leading to the induction of downstream signals, e.g. H2O2 and cytosolic Ca2+, that are required for the regulation of K+ and Na+ transporters and antioxidant defence. Consequently, K+/Na+ and ROS homeostasis of P. euphratica were maintained during a prolonged period of salt stress.
6. The physiological mechanism of PCD that induced by eATP was explored.. It has shown that eATP plays a crucial role in mediating the salinity tolerance of P. euphratica cells. However, the NaCl-induced eATP was a transient response and eATP returned to pretreatment levels after 20 min of salt and mannitol treatment. We suppose that eATP may exert adverse effects on the woody species since an excessive eATP induces PCD in animal cells. In our study, exogenously applied ATP (high dose,0.5 to 2 mM) resulted in a dose- and time-dependent reduction of viability and the agonist-treated cells displayed hallmark features indicative of PCD, such as cytoplasmic shrinkage, chromatin condensation and DNA fragmentation. A sequence of events accounting for ATP-induced PCD is proposed as evidenced using a variety of pharmacological agents. Extracellular ATP (eATP) caused an elevation of Ca2+ in the cytosol ([Ca2+]cyt), resulting from a transient influx of Ca2+ across the plasma membrane (PM) and a subsequent release of Ca2+ from the vacuole. The long-term sustained [Ca2+]cyt resulted in an evident Ca2+ uptake in the mitochondria, leading to a H2O2 accumulation therein. Noteworthy is that P. euphratica exhibited an increased mitochondrial transmembrane potential (△Ψm) and the release of cytochrome c took place without the opening of permeability transition pore over the period of ATP stimulation. Moreover, the eATP-induced increase of intracellular ATP, which is essential for the activation of caspase-like proteases and the subsequent PCD execution, was found to be correlated with the increased△Ψm. NO is implicated as a downstream component of [Ca2+]cyt but plays a negligible role in eATP-stimulated cell death. We speculate that ATP is assumed to bind P2-like receptors in the PM, leading to the induction of downstream intermediate signals because the proposed sequence of events in PCD signaling chain were terminated by an animal P2 receptor antagonist suramin.
In conclusion, at tissue and cellular levels, our data confirmed that the salt tolerance of P. euphratica is partly due to its strong ability on K+/Na+ homeostasis control. The Na+ extrusion is mainly ascribed to the strong activity of PM Na+/H+ antiport system (PM H+-ATPase and Na+/H+ antiporter) and thus, P. euphratica could restrict the radial transport of Na+ and decrease the accumulation of Na+ in the shoots and leaves. The maintenance of K+ homeostasis is mainly due to the higher activity of PM H+-ATPase, which decreased the magnitude of PM depolarization and consequently reduced the K+ loss through depolarization-activated K+ channels. At cellular level, the putative PM ATP receptors and H+-coupled ion transporters could sense osmotic and ionic effects of NaCl, respectively, and then independently activates H2O2 and Ca2+ signaling pathways. H2O2 and Ca2+ contribute to the up-regulation of K+- and Na+-related antiport system and ion channels in the PM and tonoplast, leading to a K+/Na+ homeostasis in salinised cells. In P. euphratica cells, the NaCl-induced release of ATP was hydrolysed by ecto-apyrase and the occurrence of eATP-induced PCD was avoided during a prolonged salt treatment. Finally, P. euphratica cells could survive the saline conditions under a long-term of salt stress.
引文
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