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Regulation Of Ionic And Reactive Oxygen Species Homeostasis In Seedlings Of Two Mangrove And The Relevance To Salinity

Posted on:2009-09-23Degree:DoctorType:Dissertation
Country:ChinaCandidate:N Y LiFull Text:PDF
GTID:1100360242992503Subject:Botany
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Mangroves are an assortment of tropical and subtropical trees and shrubs that have adapted to the inhospitable zone between sea and land.The most striking feature of mangroves is the ability to tolerate the seawater level of NaCl (ca. 500 mM).Moreover, some mangrove species have developed morphological alternations to adapt to saline environment, e.g. salt glands, leaf succulence, and ultra-filtration by roots. Due to the potential importance to plant breeding, much attention has been paid for elucidating salt tolerance in mangrove species. Physiological, biochemical and molecular mechanisms of salt tolerance in mangroves have been investigated, but the relationship between salt uptake, and transport regulation, ion compartmentation and salt-induced oxidative stress and the relevance to salt-tolerance is less known. Moreover, the capacity for salt tolerance remained unclear in non-secretor mangroves. Therefore, we used two typical non-secretor mangroves, Bruguiera gymnorhiza (L.)Savigny and Kandelia candel (L.)Druce in this study. In this dissertation, we illuminated the correlation of ionic and ROS regulation and salt-tolerance in the two tested species. i) Ion homeostasis. Ion relations in shoot and root tissues (root, stem, leaves and hypocotyls) and the xylem sap under increasing salinity were clarified. Compartmentation of Na+, Cl- and K+, in various leaf cells of B. gymnorhiza and K. candel were examined by means of X-ray microanalysis. In order to elucidate the salt exclusion in mangroves from the aspect of dynamic ion transport, we adopted the Non-invasive Ion-selective Microelectrode Technique to measure the real-time H+ and Na+ fluxes in root-tip cells. The species-specific differences in salt uptake, transport are apparent. ii) ROS homeostasis. We studied the effects of increasing NaCl on tissue ROS, anti-oxidative enzymes in leaves, roots and the apoplast. The ROS homeostasis control and the relevance to salinity were elucidated. We designed experiments to clarify the correlation between salt-induced oxidative stress signals and salt-tolerance in mangroves. In addition, NaCl-induced genes were preliminary characterized in the present study. In brief:1. Increasing NaCl decreased gas exchange in both species, and Pn, Gs and Tr reached the minimum at 400 mM NaCl . However, the inhibitory effects of severe salinity were more pronounced in K.candel, e.g. mean value of Pn decreased by 80% in K. candel, where as a 60% reduction was observed in B. gymnorhiza. The inhibition of light and dark reaction varied with species, especially under high saline conditions, e.g. 400 mM NaCl. (i) LCP and CCP in salinised B. gymnorhiza increased to 34μmol m-2 s-1 photon and 202μmol mol-1 CO2, while they reached 65μmol m-2 s-1 photon and 215μmol mol-1 CO2, respectively, in stressed K.candel. (ii) AQY and CE in B.gymnorhiza decreased by 33–35%, but in K. candel the extent of decline was 43-52%.2. Under non-saline conditions, K. candel contained 0.1–0.2 mmol g-1 Dry Wt Na+ in roots and leaves, respectively. While an even higher level of tissue Na+ was recorded in control B. gymnorhiza. Similarly, both species contained 0.2-0.6 mmol g-1 Dry Wt Cl- in root and shoot (stem and leaves). After exposure to 100 mM NaCl, Na+ levels in stressed B. gymnorhiza roots and leaves rapidly increased, up to 1.3-fold of that in controls, whereas Na+ in roots and leaves in of K. candel remained unchanged at 100 mM NaCl. Root and shoot Na+ in K. candel gradually increased correspondingly with the increase in salinity and with the duration of salt exposure, up to 1.6-fold of that in controls, but after exposure to 400 mM NaCl, Na+ levels in stressed B. gymnorhiza roots increased, up to 79% of that in controls. The same trend was observed in the xylem sap. High NaCl significantly increased Na+ in the xylem sap of both species, e.g. Na+ increased from 1.96 to 3.71μmol mL-1 in B. gymnorhiza and the increase of Na+ was from 2.01 to 4.36μmol mL-1 in K. candel. Therefore, B. gymnorhiza roots have a higher capacity to limit the Na+ transport from root to shoot. Salt-induced variations in Cl- differ from that of Na+. In a longer-term of salt stress (400 mM NaCl 28 days), Cl- concentration in root and leaf of K. candel increased by 25% and 42%. As compared to K. candel, NaCl-induced a higher elevation of Cl- levels in root and leaf tissues, up to 2.21-2.23 fold of that in controls, respectively. Therefore, K. candel roots remained a higher capacity to restrict the root-to-shoot transport of Cl-, especially under high salinity. NaCl-induced increase of Cl- in the xylem sap confirms the above conclusion.3. Results suggest that the salt-induced Pn reduction in the two mangrove species is correlated with the ability to exclude Na+ and Cl- from the chloroplast, rather than with the bulk leaf salt concentration. In control plants of the two mangroves, X-ray microanalysis data showed evident Na+and Cl- in the xylem vessel (vascular bundle), epidermal and mesophyll cells.Salt stress (100 mM NaCl, 7d) caused a significant rise of Na+ and Cl-in mesophyll cells of both species, but there were species-specific differences in the pattern of salt compartmentation.Vacuolar Na+ compartmentation was clearly seen in K. candel, in which the fraction of Na+ remained higher in the vacuole than in the cytoplasm and chloroplast. In contrast, there was no evidence of Cl-compartmentation in the vacuole. As distinct from K. candel, B. gymnorhiza showed vacuolar compartmentation of Cl-, relative to Na+. Noteworthy, B. gymnorhiza exhibited apparent Na+ and Cl-exclusion from mesophyll cells. Stressed B. gymnorhiza remained 23–72% lower in Na+ in all measured cell compartments, as compared to K.candel. Likewise, a lesser extent of Cl- increase occurred in the apoplast and symplast of B. gymnorhiza. NaCl (400 mM) treatment increased Na+ and Cl- concentrations in the apoplastic space, epidermal and mesophyll vacuole, but with the exception of Cl- in K. cande . In comparison, the fractions of Na+ and Cl- in the xylem vessel, cell wall and vacuole were 30-196% higher in stressed B.gymnorhiza as compared to K. candel . However, NaCl stress did not increase Na+ and Cl- concentrations in the chloroplast of the two mangroves .Vacuolar compartmentation in mesophyll was clearly seen in stressed B. gymnorhiza, in which the Na+ and Cl- concentration was higher in the vacuole than in the chloroplast . Noteworthy, B. gymnorhiza preferentially accumulated 73-94% more Na+ and Cl- in vacuoles of epidermal cells, as compared to mesophyll vacuoles . In contrast, vacuolar fractions of Na+ and Cl- in stressed K. candel remained the same as that of chloroplast, and vacuolar Na+ and Cl- in epidermis was similar to that in mesophyll vacuole regardless of treatments .4. Histochemical technique was used to localize H+-ATPase in root and leaf cells and variations of NaCl-induced ATPase were examined. Activity of plasma membrane (PM) H+-ATPase in roots and leaves of K. candel was increased by the short-term of salt treatment.. However, high activity of PM H+-ATPase was recorded in leaf cells of both control and longer-term stressed B. gymnorhiza plants. The Scanning Ion-selective Electrode Technique (SIET) was successfully used to investigate H+ and Na+ fluxes in young roots (apical zone and elongation region) of the two species. SIET data showed that salinity, short-term stress (NaCl 100 mM, 24h) or longer-term stress (NaCl 400mM, 14days), significantly increased Na+ efflux in roots of stressed plants, but B. gymnorhiza exhibited a much higher rate of Na+ efflux, as compared to K. candel. Na+ extrusion is presumably the result of Na+/H+ antiport, i.e. H+-ATPase activity was increased by salinity to create the electrochemical gradients across the PM, which is necessary for Na+/H+ antiporter to exclude more cytoplasm Na+ into the apoplast. The tendency of NaCl-induced H+ efflux indicates that salt stress has accelerated the pumping activity and the hydrolysis activity of ATPase. In accordance, Na+ levels in leaf and xylem sap of B.gymnorhiza did not increase corresponding with the increase of external saline. Therefore, the higher capacity for Na+ extrusion retained in B.gymnorhiza roots is helpful to maintain cellular and tissues ionic Homeostasis in a longer-term of salt stress.5. Increasing salinity had no significant effect on K+ , Ca2+ and Mg2 + concentrations root and shoot tissues (leaf, stem, hypocotyls) of K. candel and B. gymnorhiza. It is reasonable that the buildup of Na+ lead to increments of Na+/K+, Na+/Ca2+, Na+/Mg2+ ratios. Under the increasing salinity, the elevation of Na+/Ca2+, Na+/Mg2+ ratio in roots and leaves was significantly higher in K.candel than in B. gymnorhiza. This is resulted from the insufficient control of Na+ buildup in K.candel tissues under higher salt concentration. The concentrations of nutrient elements in the xylem sap and variations of ion relationship can directly reflect the transport of nutrient elements. The rise of Na+/K+, Na+/Ca2+, Na+/Mg2+ ratios in xylem sap of K. candel and B. gymnorhiza was mainly caused by NaCl-induced Na+ accumulation in xylem the vessels.The short-term salinity had slightly effect on Na+/K+ ratio in cell compartments of B.gymnorhiza leaves but generally increased the Na+/K+ ratio in K. candel, indicating that B. gymnorhiza was able to control celluar Na+/K+ homeostasis under salt stress.6. The concentrations of leaf malondialdehyde (MDA) remained unchanged in mangrove plants treated with different concentrations of NaCl. This suggests these plants could regulate the homeostasis of reactive oxygen species (ROS) and avoid lipid peroxidation. O2-. production rate was typically higher in roots than in leaves regardless of treatments. High salinity (400 mM NaCl) increased root O2-. by 82% and 83% in K. candel and B. gymnorhiza, respectively. An abrupt rise of H2O2 content occurred in leaf of K. candel when plants were subjected to 400 mM NaCl, although H2O2 declined after the exposure to lower salt. However, salinised B. gymnorhiza maintained a stable H2O2 level similar to control leaves despite of a NaCl increase, from 100 to 400 mM. Both mangroves up-regulated antioxidant enzymes in ASC-GSH cycle, superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), to scavenge active oxygen species, but with different patterns. K. candel rapidly triggered antioxidant defense after exposure to a lower NaCl saline (100-200 mM), whereas B. gymnorhiza maintained higher capacity to detoxify ROS at high salinity (300-400 mM NaCl). Native PAGE electrophoresis showed that the elevations of SOD and CAT in roots and leaves were coincident with a certain number of corresponding isoenzymes.7. Subcellular localization of H2O2 was carried out by CeCl3 staining. The electron-dense precipitates of H2O2 and CeCl3 were predominantly localized in cell walls and intercellular space in mesophyll of B.gymnorhiza and H2O2 production in cell walls was increased by NaCl stress (96 h). Similarly H2O2-CeCl3 precipitates were enhanced in the cell wall of stressed K.candel. We obtained xylem sap by pressure chamber to investigate salt-induced changes of ROS and anti-oxidative enzymes in the apoplast. Results showed that the salt ion accumulation in the apoplast of both mangrove species induced ROS (O2-. and H2O2) buildup in a long-term salt stress. Anti-oxidative enzymes (SOD, CAT, APX) was increased corresponding to the salt-induced ROS, especially in B.gymnorhiza. Therefore, up-regulation of anti-oxidative enzymes in B.gymnorhiza was able to prevent salt-induced excess ROS accumulation and the subsequent oxidative injury, which contributing to membrane stability maintenance.8. Both mangrove species could sense the initial salt stress, and increase ABA content in root and leaves, correspondingly. In comparison, B. gymnorrhiza maintained higher levels of ABA leaves under high salinity (400 mM). Xylem-fed ABA reduced leaf Pn, Gs and Tr, but led to a significant increases in the production H2O2 and antioxidant enzymes in the two mangroves. H2O2 levels and antioxidant enzymes in leaves of the two species were elevated by xylem-feeding H2O2. Using ABA synthesis inhibitor, tungstate (sodium form), significantly decreased the NaCl-induced increase in ABA and antioxidant enzymes. These results suggest that NaCl induces an ABA accumulation in mangroves, which accelerates H2O2 generation and antioxidant enzymes. The up-regulation of antioxidative defense is helpful to scavenge salt-induced ROS. K.candel was able to incease ABA after the onset of salt stress, rapidly initiated anti-oxidant defense in root and leaves to reduce ROS at an early stage of salt stress. In comparison, B. gymnorrhiza leaves maintained higher levels of ABA and a greater capacity to detoxify ROS at high salinity.9. AUTOLAB Kit were employed to extract total RNA from leaves of K. candel treated with NaCl concentration ranging from 100 to 400 mM (salinity started from 100 mM and increased stepwise by 100 mM, reaching 400 mM NaCl for 24h). The object was to identify genes that were differentially expressed in response to salt stress. The electrophoresis analysis showed that there were apparent integrate bands of 28S and 18S rRNA. Twelve clones were chosen randomly from the cDNA library. By means of PCR, 10 PCR fragments were obtained, ranging from 0. 25 to 1kb. ESTs similarity analysis based on BLASTx software was finished by comparing sequences in non-redundance database of GenBank. Most of ESTs had higher homology with Hsp of potato and tomoto plants. In conclusion, there are genotypic differences in salt tolerance between the two typical non-secretor mangroves, B. gymnorhiza and K. candel:(1) K. candel could restrict the entry of salt, which is necessary to avoid abrupt increase of salt in shoots. Moreover, K. candel is sensitive to lower soil salt and synthesize ABA, rapidly initiates antioxidant defense to scavenge reactive oxygen species by, at least in part, components of the ASC-GSH) cycle, e.g. SOD, APX, CAT and GR.(2) B. gymnorhiza took up much salt ions at high salinity, but the effective vacuolar salt compartmention in mesophyll cells, and the preferential accumulation of Na+ and Cl- in epidermal vacuoles may benefit B. gymnorhiza plants to reduce ROS production in the mesophyll. Higher salinity up-regulated ABA and CaM, as well as antioxidant enzymes, which limited ROS level in leaves, roots and xylem sap of B. gymnorhiza. The critical balance between ROS production and ROS detoxification is remained under salt stress. We concluded that B. gymnorhiza is more salt resistant than K. candel.
Keywords/Search Tags:Mangrove, salt resistance, photosynthesis, Non-invasive Ion-selective Microelectrode, ion compartmentation, oxidative stress signal
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