Salt-induced Oxidative Stress And The Relevance To Salt Tolerance In Poplar

Populus euphratica Oliv. 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, but it's potential application value is not developed abundantly. Recently, physiological, biochemical and molecular mechanisms of salt tolerance have been investigated in P. euphratica, but the relationship between salt-induced oxidative stress and salt-tolerance is less known. Moreover, salt-induced oxidative stress has received much attention. In order to elucidate mechanism of reactive oxygen species (ROS) regulation and the relevance to salt-tolerance in woody plants, we used salt-tolerant P. euphratica and salt-sensitive P. popularis and P. cv I-214 to investigate the effects of increasing NaCl on tissue salt concentration, ROS, anti-oxidative enzymes, malondialdehyde (MDA) and membrane permeability (MP) in leaves, roots and the apoplast. In addition, we designed experiments to clarify the correlation between salt-induced oxidative stress signal and salt-tolerance in poplars.1. In a longer-term of salinity, CO₂ assimilation in P. popularis was severely reduced whereas stressed P. euphratica maintained a relatively higher and constant level of Pn. Pn-Ci curves showed that salt stress (12 days) reduced CO₂ saturation point (CSP), CO₂ saturated Pn (CSPn), and carboxylation efficiency (CE), but increased CO₂ compensation point (CCP) in the two genotypes. Similarly, salinity lowered light saturation point (LSP), light saturated Pn (LSPn), and apparent quantum yield (AQY) in both genotypes but the inhibitory effect of NaCl on light reaction was more pronounced in P. popularis, as compared to P. euphratica. Chlorophyll a fluorescence data indicated that a longer-term of salt stress (12 days) exhibited a marked influence on fluorescence parameters of P. popularis in both dark- and light-adapted states: (i) NaCl inhibited the maximal efficiency of PSII photochemistry (Fv/Fm) due to the salt-induced increase of Fo (the minimal fluorescence) and the marked decline of Fm (the maximal fluorescence); (ii) salinity decreased coefficient of photochemical quenching (qP) but markedly elevated coefficient of nonphotochemical quenching (qN) in the light-adapted state. In contrast, there were no corresponding changes of chlorophyll a fluorescence in salinised P. euphratica. This indicated less effect of same salt stress on photosynthesis in P. euphratica is than P. popularis.2. Inhibition of leaf photosynthesis is related to membrane damage caused by salt build-up in the long-term salt stress. Results show that salt ion accumulation in leaves, especially in chloroplast, inhibited photosynthesis of P. popularis. The increase of MP and MDA was resulted from a salt buildup in leaves, which leads to membrane peroxide and in ion leakage. In contrast, P. euphratica maintained stability of membrane system under salt stress and salinity did not cause membrane peroxidation since less salt ion accumulated in P. euphratica leaves.3. Results show that the acceleration of membrane peroxidation was associated with ROS regulation. At the beginning of salinity, P. euphratica can alleviated NaCl-induced water stress and limited shoot-to-root transport of salt ion by inducing abscission of yellow leaves in lower shoot, Moreover leaf abscission was associated with a increase of ethylene synthesis induced by superoxide radical (O₂^-·) upregulation. P. euphratica significantly increased anti-oxidative enzymes (ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) in leaves of lower shoot to control levels of O₂^-·and H₂O₂, thus avoiding oxidative damage and maintaining integrality of membrane system. In contrast, P. popularis is not able to sense salt stress and make corresponding changes for salt adaptation. After exposure to salt stress, a great buildup of Na+ and Cl- restraining photosynthesis induced a significant increase of ROS in leaves and xylem sap of P. popularis despite a notable increase of anti-oxidative enzymes, which resulted in leaf-injury ultimately. Unlike P. popularis, P. euphratica could effectively prevent salt-induced ROS accumulation by up-regulating anti-oxidative enzymes although there was a great buildup of salt ion in leaves and xylem (especially Na), which is favorable to avoid oxidative damage and maintain membrane integrity. Accordingly, that the capacity for anti-oxidative stress plays a important part in salt tolerance in poplars.4. We investigated the effect of a long-term salt stress on ROS and anti-oxidative enzymes activities in roots. Results show that P. euphratica root could rapidly sense the initial salinity and reduce salt-induced oxidative damage by up-regulating anti-oxidative enzymes; while in a long-term salt stress, P. euphratica could effectively control ROS levels by maintaining higher level anti-oxidative enzymes system, which is benefit to avoid oxidative damage and maintain membrane stability, the salt ion absorption and transport were consequently reduced. However, P. popularis was not able to up-regulate anti-oxidative enzymes after the onset of salt stress. The salt-induced decline of anti-oxidative enzymes in P. popularis, leads to oxidative injury of membrane system, which causing salt ion accumulation.5. We obtained xylem sap by pressure chamber to investigate salt-induced change of ROS and anti-oxidative enzymes in the apoplast. Cellular H₂O₂ location was determined by cytochemistry staining. Results showed that the salt ion accumulation in the apoplast of P. popularis induced ROS (O₂^-·and H₂O₂) buildup in a long-term salt stress. The increased anti-oxidative enzymes was not able to remove ROS injury, leading to salt damage in P. popularis leaves. Although salt ion buildup took place in P. euphratica leaves, activities of anti-oxidative enzymes were inceased correspondingly to prevent salt-induced excess ROS accumulation and avoid oxidative injury, which contributing to membrane stability maintenance.6. Using ABA synthesis inhibitor, we founded that salt induced ABA signal transduction was associated with salt tolerance in P. euphratica. Salt-tolerant P. euphratica could sense the initial salt stress, incease ABA content in leaves and induced stomata close, which alleviates salt-induced osmotic stress and limit salt absorption and the root-to-shoot transport. Moreover, ROS participated in ABA-induced stomata close and up-regulation of anti-oxidative capacity. In contrast, salt-sensitive P. popularis could not make rapid adaptations to saline conditions.In conclusion, salt tolerance of P. euphratica may be a consequence of salt-induced anti-oxidative stress. P. euphratica induced leaves abscission to reduce water loss at the beginning of salt tolerance. In the long term of salt stress, P. euphratica limited ROS level in leaves, roots and apoplast to avoided oxidative injury, which is benefit to membrane system stability. Noteworthy, salt-induced oxidative stress signal played an important role in salt-tolerance of P. euphratica, which caused stomata closure and reduced transpiration and oxidative injury.