| Soil salinity together with secondary salinisation are worldwide environmental issues that causing land productivity degradation, ecological environment deterioration, crisis of food and ecological security. To clarify salt-tolerance mechanism and breeding new salt-tolerant plant species are important for improving soil salinity. Mangrove plant, thriving in the intertidal zone, is another model plant for plant salt-tolerance mechanism research in addition to typical terrestrial plants. Elucidating salt-tolerance mechanism of mangrove plant can provide theoretical guidance and technical support for afforestation in coastal saline areas.Maintenance of K+/Na+homeostasis is crucial for plants adapting to saline environments. Salt-stress signaling moleculars play an important regulationg role in maintaining K+/Na+homeostasis. At present, the mechanisms regulating ion homeostasis by signaling moleculars in these non-secretor mangroves are largely unknown since this topic has been less and non-systematicly investigated. Therefore, in this study, using seedlings of Bruguiera gymnorrhiza (L.) Savigny and Kandelia candel (L.) Druce, we examined the NaCl-induced ion accumulation in different tissues, and the effects of signaling moleculars (H2O2, NO, CaCl2) and plasma membrane Na+/H+antiporter system on K+/Na+homeostasis were investigated in the two species. Moreover, KcTIPl, encoding tonoplast intrinsic proteins TIP1, was cloned from K. candel and transferred to tobacco plant. The aim is to clarify the role of KcTIPl in osmotic regulation and salt tolerance of plant. The main results and conclusions are as follows:1. Using seedlings of the two typical non-secretor mangroves as experimental material, the response of tissue of Na+,Cl",K+and Ca2+to different growing environment and NaCl saline were tested.(1) The accumulated amount of salt ions and nutrient elements of tissues from the both species grown in natural environment was higher than in greenhouse. However, the distribution of Na+, Cl", K+were consistent in organs of the both species irrespective of growing environments. Accordingly, greenhouse-grown mangrove plants were used in our study instead of naturally-grown ones.(2) For salt ion accumulation, Na+concentration in root tissue of K. candel was increased after24h of100mmol NaCl. High salinity (400mmol/L NaCl/28d) enhanced salt ion accumulation in the two species. However, there were species differences in the salt accumulation between the two species.(A) K. candel exhibited a higher increase of Na+content in the root, stem and leaf tissues than B. gymnorrhiza. But the hypocotyl of B. gymnorrhiza accumulated a higher Na+.(B) Compared to K.candel, B. gymnorrhiza exhibited a higher increase of Cl" in root and stem, but with a lower Cl-concentrations in the hypocotyl and leaves. The results indicate that the hypocotyl of B. gymnorrhiza has a high capacity for Na+sequestraton. Compared to B. gymnorrhiza, K. candel retained higher Cl-in hypocotyl tissue.(3) For nutrients accumulation, high salinity caused a decline of nutritional level in roots of both species (except for Mg+in K. candel). This reveals that part of nutrients was replaced by Na+.(4) For salt ions and nutrients homeostasis, K. candel has a higher capacity to maintain K+/Na+homeostasis under short term salt treatment. However, B. gymnorrhiza has a higher capacity to maintain K+/Na+homeostasis after long term salt exposure.2. Using young roots of seedlings of B. gymnorrhiza and K. candel, we measured Na+、H+、Cl-and K+fluxes by SIET.(1) ST (100mmol/L NaCl,24h) and LT (200mmol/LNaCl,7d) treatment resulted in a Na+efflux and a correspondingly increased H+influx in roots of both species. 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.(2) ST and LT treatment also induced Cl-efflux from roots of both species and Cl-efflux was higher than Na+efflux. We speculate that Cl-transport from root to shoot was restricted and there are Cl-resources in mangrove roots. In addition, Cl-efflux was higher in root of B. gymnorrhiza, compared to K. candel, indicating that B. gymnorrhiza has higher capacity to exclude Cl-.(3) NaCl treatments (ST and LT) caused an evident K’efflux in the two species, which was significantly reduced by the K+channel blocker TEA. This result indicates that salt-induced K+efflux is mediated by the depolarization-activated channels, e.g., KORCs and NSCCs. B. gymnorrhiza roots exhibited a typically lower K+efflux than K. candel under ST and LT salinity. The capacity for B. gymnorrhiza to retain K+is likely the result of its high activity of PM H+-ATPase.(4) A more pronounced Na+efflux was observed in B. gymnorrhiza under LT treatment, and K+efflux was more lower in B. gymnorrhiza than in K. candel under both ST or LT treatment, indicating that B. gymnorrhiza has a higher capacity in maintaining K+/Na+homeostasis under salinity.(5) Mannitol treatment (170mmol/L Mannitol, water potential is equal to100mmol/L NaCl) did not induce obvious changes of Na+, H+, Cl-, K+fluxes from B. gymnorrhiza roots, indicating that the response of B. gymnorrhiza to high salinity was ion specific. However, a significant Na+efflux was observed from mannitol-treatment root of K.candel. This implies that the salt-induced Na+efflux in K. candel root was partly from osmotic effect.3. Using young roots of seedlings of B. gymnorrhiza and K. candel, salt-altered flux profiles of Na+and K+from roots of both species and the effects of H2O2, NO, Ca2+and PM Na+/H+antiport system on root ion fluxes were examined. However, there were significant differences in the regulation of ionic homeostasis between the two tested mangrove species.(1)(A)Exogenous H2O2and Ca2+application markedly increased the Na+efflux and limited K+efflux induced by ST treatment. Of note, NaCl-induced Na+efflux was inhibited by DPI (the inhibitor of NADPH oxidase), DMTU (ROS scavenger), catalase and LaCl3(inhibitor of Ca2+channel of PM), However, K+efflux was increased by these inhibitors. Results indicate that H2O2and Ca2+positively mediated K+/Na+homeostasis under saline environment.(B) The salt treatment induced an evident H2O2efflux, which was inhibited by DPI, DMTU, amiloride and orthovanadate. This reveals that NADPH oxidase and PM Na/H+antiporters were involved in salt-induced H2O2burst. The salt treatment also induced an evident Ca+influx, which was inhibited by , amiloride and orthovanadate. This reveals that PM Na+/H+antiporters were involved in salt-induced Ca2+influx mediated by Ca2+channel of PM.(C) NaCl treatment caused an obvious influx of Ca2+in roots of B. gymnorrhiza, which was enhanced by H2O2but suppressed by DPI. Exogenously applied H2O2caused an influx of Ca2+, which was inhibited by LaCl3. This reveals that Ca2+was involved in H2O2-mediated Na+/K+homeostasis.(2) The experiment mentioned above were carried on K. candel, but different pattern was observed. Results indicate that H2O2has a positive effect on K+/Na+homeostasis, but effects of Ca2+remains further research.(3) Under ST treatment, K+loss inhibited by SNP (100μmol/L, a NO donor) was only seen in K. candel.4. Using young leaves of K. candel as material, the KcTIP1gene was amplified by PCR and its expression vector was constructed. Then it was integrated into tobacco plants via Agrobacterium-mediated transformation systems. Transgenic plants were selected by kanamycin and verified by genomic PCR and RT-PCR analysis. Two transgenic lines as well as the wild-type were treated with NaCl (150mmol/L,7d). After salt treatment, fresh weight of root, stem and leaf tissues, net photosynthetic rate and transpiration rate, were measured in KcTIP1-transgenic and wild-type tobaccos. In addition, leaf Na+and K+were quantified and young roots with apices of1.0cm were used for steady flux measurements of Na+and K+. In our study, analysis of KcTIP1amino acid sequence indicates that KcTIP1contains the MIP family signature sequence SGGHVNPAVT and two highly conserved sites of NPA (Asn-Pro-Ala) protein. RT-PCR analysis shows that the KcTIP1gene was overexpressed in two transgenic lines (TR1and TR2). After salt treatment, the fresh weight, net photosynthetic rate and transpiration rate were significantly higher in KcTIP1-transgenic lines than in the wild-type plants. In leaf tissues, Na+content of KcTIP1-transgenic plants was highly accumulated compared to the wild-type; however, the loss of K+was less in KcTIP1-transgenic tobacco leaf tissue than in the wild-type. SIET data showed that salt treatment resulted in a higher influx of Na+and K+in root of KcTIP1-transgenic type, compared to the wild type. In conclusion, KcTIP1has similar protein structure and zymologic function with other Aquaporins. The KcTIP1overexpression improved salt tolerance of tobacco plants by increasing photosynthetic rate, transpiration rate and improving osmotic potential. In addition, we speculate the KcTIP1overexpression also enhances the salt tolerance by regulation of K+/Na+homeostasis. |
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