| Limonium bicolor (Bag.) Kuntze is a perennial herb belonging to the Plumbaginaceae family, distributing at saline soil areas in the coastal and grasslands. Limonium bicolor is a typical recretohalophyte as well as a medicinal plant. In this study, a variety of biological indicators of L. bicolor were measured under salt-stress to explain the salt-tolerance mechanism, including botanical characters, physiological and biochemical responses,zymoproteins, chemical components and regulatory genes expression. Changes of main medicinal ingredients in L. bicolor under salt stress was observed by the qualitative and relative quantitative analysis in control and salt treatment. The regulation rules for ingredients in L. bicolor by saline environment was found. The results can provide a reference for further understanding of the tolerant mechanism of halophytes and the effect of environment on plant secondary metabolites. It has great significance of L. bicolor in use of saline land as halophytes and in medicine as traditional Chinese medicinal materials. The specific research results are as follows.1. Effects of salinity on botanical traits, physiological and biochemical indicators and chemical compositions in L. bicolorIn this study, L. bicolor was treated with NaCl solutions with concentrations of 0 mM,100 mM, 200 mM and 300 mM. In order to ascertain the physiological mechanisms of L.bicolor to defend salt stress, a series of botanical characteristics, physiological and biochemical indexes and changes of chemical compositions were determined. In present study, fresh weight and dry weight of leaves and roots were inhibited firstly at all of the salt treatments and then gradually increased in 100 and 200 mM as time changed. On the 21 d after treatment, the maximum of dry weight of both the leaves and roots appeared at 200 mM and the minimum were in 300 mM. With the change of time, tissue water content gradually increased in 100 mM NaCl treatment while decreasing in other treatment groups.PN value was significantly higher at 100 and 200 mM and lower at 300 mM than that of control. Meanwhile, the soluble protein content in leaves was also up regulated in 100 and 200 mM NaCl. These results indicated that 100 and 200 mM NaCl could induce positive effects in synthesis of biomass in L. bicolor while 300 mM NaCl could inhibit the synthesis.Besides, the root activity decreased with the increased salt concentrations. Na+ content,MDA content and EL value in leaves were all obviously increased at 300 mM NaCl. It suggested that excess Na+ accumulating in leaves under this concentration resulted in the injury of cell membrane. It was found that the number of salt glands on leaf surface changed significantly with increased concentrations of NaCl. The number of salt glands on both adaxial and abaxial surface of L. bicolor leaves increased considerably as the salt concentration rose and then significantly decreased at 300 mM NaCl with the peak appeared at 200 mM NaCl. This may be the direct reason of the changes of Na+ content in leaves. SOD activity firstly increased and then decreased as the increased salt concentration with a maximum at 200 mM NaCl. However,POD activity decreased gradually as the salt concentration increased and CAT activity showed no obvious difference between different NaCl concentrations. NaCl stress induced various effects on the accumulations of total phenolic and flavonoid in L. bicolor. The contents of total phenolic and flavonoid decreased with the increased salt concentrations and reached the minimum at 300 mM in leaves and at 200 mM in roots, respectively. Accumulation of gallic acid and myricetrin changed obviously under different NaCl concentrations. The content of gallic acid was enhanced by 200 mM NaCl in leaves and reduced by 200 and 300 mM NaCl in roots. The content of myricetrin also showed the maximum at 200 mM NaCl while fewer content in all of the salt treatments compared to the control. Results showed that salt treatment affected the accumulation of some chemical compositions in L. bicolor. In conclusion, changes of biomass, leaf Na+ content, electrolyte leakage, MDA content and photosynthetic characteristics showed the similar trend in L.bicolor under salt-stress,which exhibited good adaptability at 200 mM NaCl and inhibition at 300 mM NaCl. When NaCl concentration was lower than 200 mM, Na+ content in leaves was kept stable, autologous defense capabilities was enhanced and oxidative damage was reduced by improving photosynthetic efficiency, antioxidant enzyme activity, synthesis and accumulation of anti-oxidative substances such as flavonoids and phenolic acids. Finally, we speculated that the salt-tolerance mechanism of L. bicolor involved in the increased number of salt glands,enhanced enzyme activities, and the accelerated accumulation of secondary metabolites.2. Effects of salinity on proteomics in L. bicolorIn this study, differentially expressed proteins were obtained from leaves and roots of L.bicolor after 200 mM NaCl treatment (0 h, 48 h) by using the two-dimensional electrophoresis and MALDI-TOF-TOF-MS technologies. In leaves, 38 unique proteins differentially expressed were identified. These proteins were mainly involved in Carbohydrate transport and metabolism (4%), Energy production and conversion (8%),Amino acid transport and metabolism (26%), Lipid transport and metabolism (4%),Secondary metabolites biosynthesis, transport and catabolism (8%), Inorganic ion transport and metabolism (8%), Translation, ribosomal structure and biogenesis (4%),Posttranslational modification, protein turnover, chaperones (30%), Cytoskeleton (4%),General function prediction only (4%) and unclassified proteins? In roots, 41 unique proteins differentially expressed were identified. These proteins were mainly involved in Carbohydrate transport and metabolism (20%), Energy production and conversion(10%), Amino acid transport and metabolism (17%), Inorganic ion transport and metabolism (7%), Translation, ribosomal structure and biogenesis (7%), Posttranslational modification, protein turnover, chaperones (13%), Cytoskeleton (3%), Transcription (3%),Defense mechanisms (3%), Signal transduction mechanisms (3%), Cell wall/membrane/envelope biogenesis (7%),Intracellular trafficking,secretion, and vesicular transport (7%) and unclassified proteins. Taken together, the synergistic changes in proteome including photosynthesis, carbohydrate and energy metabolismes, secondary metabolites biosynthesis and so on enabled L. bicolor to tolerate salt stress. This study provides important information for further investigation of the salt -tolerance mechanism in plants.3. Effects of salinity on chemical compositions in L. bicolorIn this study, UPLC / Q-TOF-MS techniques were used to the qualitative and quantitative analysis of the main chemical components in L. bicolor under salt treatment. 11 major compounds had been identified from the leaves and roots of L. bicolor. They were triethyl citrate(1), gallic acid(2), vanilla acid(3), vanilla acid derivative(4), vanilla acid-glucoside/gal(5), N-(2-carboxyethyl) iminodiacetic acid(6), epigallocatechin gallate(7),myricetin-3-O-?-D-glucoside(8), myricetin-3-O-?-L-rhamnosides(9), quercetin-3-O-a-L-rhamnosides (10) and myricetin-3-O-?-D-(6"-gallic acid)-galactosidase (11). Types of compounds were similar between roots and leaves but the contents of those compounds were significantly different. The compound 6 appears only in the leaves and the compound 4 appears only in roots. Contents of compound 1, 3, 4, 7, 8 and 9 were significantly higher in roots than that of leaves before salt treatment. And contents of compound 2, 5, 10 and 11 were significantly higher in leaves than that of roots. Contents and distributions of compounds changed obviously in leaves and roots after 200 mM NaCl treatment. Contents of compound 1, 3, 6 and 7 were decreased when compared with the control in leaves.Meanwhile, contents of compound 2, 5, 8, 9, 10 and 11 were increased when compared with control. Contents of compound 1, 2, 3, 5, 7, 9,10 and 11 showed significant difference.Contents of compound 1, 2, 3, 4, 5, 8 and 11 were decreased when compared with the control in roots. Meanwhile, contents of compound 7 and 9 were increased when compared with control. Contents of compound 1,2,3,4,5, 7, 8 and 9 showed significant difference. It was also found that the distribution of compounds in the leaves and roots was also affected by salt. After the 200 mM NaCl treatment, contents of the compound 1 and 7 were increased in roots when compared with those in leaves and the content proportions of compound 2, 3, 5, 8 and 10 were decreased in roots when compared with those in leaves. It was concluded that NaCl stress induced the changes in contents and redistribution of flavonoids and phenolic acids in different tissues of L. bicolor. Myricitrin compounds changed significantly suggesting that it might play a role in the antioxidant defense system when L. bicolor under salt stress.4. Cloning of CHS fragment and expression analysis of CHSFlavonoids are one of the most important chemical compositions of L. bicolor and non-enzymatic antioxidants under salt stress in L. bicolor. In order to verify the interaction of salinity on the expression of the key enzymes CHS gene in flavonoid biosynthetic approach in L. bicolor, total RNA of control and salt treatment were extracted for cloning fragment and analysis of expressions of CHS gene. Results showed that expression level of CHS gene was significantly increased when compared with the control in leaves but decreased in roots at 48 h, 200 mM NaCl treatment. This result indicated that CHS gene of L. bicolor was sensitive to NaCl and were consistent with the findings of changes in chemical compositions induced by NaCl. |
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