Effects And Mechanisms Of Nitric Oxide On Regulating Heavy Metal Stress In Two Landscape Herbaceous

During the last decades, environmental (heavy)-metal pollutants has increased dramatically due to industrial and agricultural activities, increasing the potential risk to urban ecology and human health. Therefore, repairing and governancing of metals pollution has become an increasingly urgent problem worldwide. Fortunately, metal-polluted envriomnent remediation by phytoremediation technology (because of its safety and low-cost characteristics) is the cutting edge of plant nutrition and environment bioremediation research. However, most of hyperaccumulators for phytoremediation are only suitable for theoretical research of resistance mechanism which due to their slower growth, lower metal-transportion, poor landscape, and geographical restrictions, and are not suitable for soil remediation in a large area of metal-contaminated. Accordingly, if some important herbs with economic and ecological values that used in landscape gardens could be successfully selected as the repair materials of metal-contamination soil, which will be a new program for the phytoremediation of metal-pollution. Based on the above background, in the work, two important metal-resistant landscape plants [ie. white clover(Trifolium repens) and periwinkle(Catharanthus roseus)] are detected for the effects of soil metals (Cd or Cu) on the levels of physiological, biochemical, cellular and molecular after three different time (45 d,60 d and 80 d) exposure by using metabolomics, Western blot, fluorescence microscopy, proteomics (iTRAQ) and other methods. In addition, we also detected and discussed the regulatory mechanisms of applying or spraying exogenous NO (in the term of sodium nitroprusside/SNP) in the metal-induced plants. In our present study, four conclusions are obtained as below:1. Trifolium repens is an important and widespread forage legume and cover crop because of its outstanding forage quality and high animal-feeding value, also has a high agronomic value because of its high nitrogen-fixing ability. The results indicated that the treatments with higher Cd concentrations (>50.00 mg·kg-1) significantly inhibited white clover biomass and C, N, P and K accumulation, as well as altered their distribution patterns, while the treatment with lower Cd concentration (<25.00 mg·kg-1) had no significant effects. By the detection of western blot and enzymes activity showed,50.00 mg·kg-1 soil Cd after 45 days treatment significantly improved the activity and protein of monodehydroascorbate reductase (MDHAR) and the content of total thiols (TT), but declined the activities and proteins of superoxide dismutase (SOD) isoenzymes (CuZn-SOD and Mn-SOD) and CAT, and the levels of ascorbic acid (ASC), reduced glutathione (GSH), docosahexaenoic acid (DHA), oxidized glutathione (GSSG) and non-protein thiol (NPT) in leaves. In terms of the root system, Cd stress increased the activity (and/or protein) of SOD, TT, ASC, but significantly reduced the levels of glutathione reductase (GR), guaiacol peroxidase (GPX), DHA and GSSG. Meanwhile, the study showed by the Native-PAGE analysis that, Cd treatment significantly reduced the protein activity of root CuZn-SOD, enhance the protein activity of Mn-SOD protein activity. Moreover, using the semi-quantitative RT-PCR validation showed that Cd treatment reduced the expression of CAT and CuZn-SOD, but raised the protein expression of APX, Fe-SOD, Mn-SOD and GR in the roots. Besides,50.00 mg·kg-1 Cd stress significantly enhance the levels of ROS [H2O2 detected by DCF-DA fluorescence) and O2·- detected by DHE fluorescence] and MDA in the shoots and roos of white clover plants, and also increased the production of NO in vivo (35%, using a DAF-2DA fluorescence) in leaves, but significantly reduce the NO generation and fluorescence intensity in taproots and lateral roots (> 50%, DAF-2DA). Thus, to a certain extent, white clover has stronger tolerance to Cd contamination. These results suggested that white clover has some potential applications in the remediation of Cd -contaminated soil, which also provide some scientific reference for forage security monitoring and evaluation.2. The results obtained in this work indicate that different concentrations of SNP (i.e. 0,50,100,200 and 400 ?mol·L-1) applied exogenously, particularly at lower doses (50 ?mol·L-1), can effectively depleted the detrimental effects on white clover growth when compared with the treatment of 50 mg·kg-1 soil Cd after 45 days exposure, likely by reducing oxidative damage, modulating mineral absorption, and re-establishing the levels of ATPases. Furthermore, with respect to 50 mg·kg-1 Cd treatment for 80 days, the application of 50 ?mol-L-1 SNP markedly enhanced plant dry-weight (DW), chlorophyll content, and NO generation (and fluorescence intensity; DAF-2DA) in leaves and roots, improved the uptake of minerals [ie, potassium (K), calcium (Ca), magnesium (Mg), iron (Fe) and copper (Cu)], enhanced the metabolism of ROS scavenging system, and reduced the absorption of tissu Cd ions in plant tissues, thereby offsetting the Cd-induced toxic. Even so, the alleviating effect of SNP on plant growth was counteracted by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide, a NO scavenger) and 1-NAME (NG-nitro-L-Arg-methyl ester, a NO-synthase inhibitor) and was not observed with SNP analogues (sodium nitrite/nitrate and sodium ferrocyanide), suggesting that the protective roles of SNP are related to the induction of NO. To the best of our knowledge, our findings are the first to demonstrate clearly that NO could regulated the acitivities of ATPase (PM-H+-ATPase and TV-H+-ATPase) and AMPase (PM-5'-AMPase) in proton pump, enhanced the contents of jasmonic acid (JA) and pro line (PRO) in shoots and roots of Cd-treated plants. These results suggest that NO may improve the Cd tolerance by eliminating oxidative damage, re-establishing ATPase activity, and maintaining hormone equilibrium.3. Madagascar periwinkle, a highly studied and exploited medicinal plant and a popular ornamental plant, contains more than 100 monoterpenoid indole alkaloids (MIAs) that are used for the treatment of both malignant and non-malignant tumors and platelet (associated) disorders. All parts of the plant are credited with hypoglycemic properties and are used to treat diabetes. Meanwhile, the anticancer alkaloids vincristine and vinblastine are primarily present in the shoots (leaves), and the antihypertensive alkaloid ajmalicine is found in the roots. Periwinkle is also a good source of antioxidants for scavenging free radicals, and antioxidant metabolism under salt stress, water deficits, hormone induction and growth regulation has been studied in these plants. In the work, the effects of 30 mg kg-1 copper/Cu,50?mol·L-1 SNP and their combination (Cu+SNP) on the metabolism of the medicinal plant Catharanthus roseus were investigated 60 days after planting. Cu markedly decreased plant growth, soluble proteins and endogenous NO and disrupted mineral equilibrium and proton pumps. In addition, Cu increased Cu2+ accumulation, stimulating reactive oxygen species burst. However, the SNP treatments reversed most of these effects. However, at the root level, SNP improved nearly all of the selected amino acids (AAs); only serine (SER) and methionine (MET) did not exhibit their peak values. In contrast, the Cu+SNP treatment decreased their levels (arginine/AGR, aspartate/ASP, MET and SER decreased slightly compared to the SNP treatment). Relative to the control, the phenylalanine ammonia-lyase (PAL) activities in the shoots and roots were decreased by the Cu treatment but increased by the SNP treatment (+19% and+26% of the control, respectively). Meanwhile, the Cu+SNP treatment elevated the PAL activity above that observed for the Cu treatment. A similar trend was observed for the total soluble phenols. Among the 11 phenolic acids, the SNP in the Cu+SNP treatment had a favorable effect on the observed quantitative changes. Interestingly, the SNP and Cu+SNP treatments had a higher stimulatory effect than the Cu treatment (for salicylic, syringic and vanillic acids in shoots and salicylic, vanillin, p-OH-benzoic aldehyde and protocatechuic/aklehyde in roots). Nevertheless, the stimulatory effects on the sum of the acids in the shoots induced by the SNP and Cu+SNP treatments were not higher than that induced by the Cu treatment. However, the opposite was true for the roots. On the other hand, the accumulation of vincristine and vinblastine in the leaves was reduced by Cu, and their maximum values were observed in the SNP treatment (+38% and+49% of the control, respectively). The less-pronounced effect of the SNP treatment compared to the Cu treatment led to the observation of the same effect in the Cu+SNP treatment. Similarly, the total alkaloid content in the plants was increased by the SNP (ca.1.5-fold) and Cu+SNP treatments. However, Cu exposure resulted in an apparent depletion effect. Unexpectedly, the ajmalicine content in the roots was unaffected by the four treatments (P>0.05). This study provides the first evidence of NO having strong protective properties against the negative effects induced by Cu and discusses potential signaling mechanisms.4. Besides, we also investigated the impacts of 50 ?mol·L-1 spraying exogenous NO on the proteomics of Catharanthus roseus leaves grown in pot culture with 30 mg·kg-1 Cu for 60 days exposure by using the latest iTRAQ technology. Studies have shown that, on a 10% confidence level,19,537 unique peptides,2,560 expressed proteins that removed redundant protein, and 114,558 spectrogram and 32.2% in spectrum utilization rate were identified. Moreover,1,370 trusted protein, less than 1% in Global FDR from Fit, in the whole comparison group ([B-A], [C-A], [C-B], [D-A], [D-B] and [D-C] groups) were obtained, which was analyzed based on the specific peptide segment data of 4 iTRAQ protein quantitative selection. And,181 differentially expressed proteins in Dynamics were obtained according to different proteins screening criteria. Among them, 181 differentially expressed proteins in B-A group (i.e. [Cu]?[Control],55 up- and 88 down-regulated proteins),57 differentially expressed proteins in C-A group ([SNP]?[Control],24 up- and 32 down-regulated proteins),76 differentially expressed proteins in [D-A] group ([Cu+SNP]?[Control],27 up- and 48 down-regulated proteins), 92 differentially expressed proteins in [C-B] group ([SNP]?[Cu],60 up- and 31 down-regulated proteins),97 differentially expressed proteins in D-B group ([SNP]?[Cu],55 up- and 41 down-regulated proteins), and 31 differentially expressed proteins in [D-C] group ([Cu+SNP SNP]?[Cu],12 up- and 18 down-regulated proteins) were obtained. Furthermore, we also analyzed the Protein changes of [B-A], [A-C] and [D-B] groups by the analysis of Gene Ontology/GO (Biological Process (BP), Cellular Localization (CC), Molecular Function (MF)], KEGG pathway (Kyoto encyclopedia of Genes and Genomes) and Protein-Protein Interaction (PPI).? In [B-A] group,1,158 protein involve in the biological process (BP), and 587 protein are enriched in a significant (at P<0.05) level; 156 protein involve in the cellular localization (CC), and 85 protein are enriched in a significant level; and 324 protein involve in the molecular function (MF), and 175 protein are enriched in a significant level. In addition,71 protein involve in the KEGG pathway, and 15 protein are significant (at P<..05) features.? In [D-B] group,1,065 protein involve in the biological process (BP), and 530 protein are enriched in a significant (at P<0.05) level; 158 protein involve in the cellular localization (CC), and 85 protein are enriched in a significant level; and 270 protein involve in the molecular function (MF), and 129 protein are enriched in a significant level. In addition,55 protein involve in the KEGG pathway, and 12 protein are significant (at P<0.05) features.? In [D-B] group,905 protein involve in the biological process (BP), and 491 protein are enriched in a significant (at P<0.05) level; 133 protein involve in the cellular localization (CC), and 76 protein are enriched in a significant level; and 258 protein involve in the molecular function (MF), and 117 protein are enriched in a significant level. In addition,54 protein involve in the KEGG pathway, and 14 protein are significant (at P<.0.05) features.Overall, NO involved invarious physiological activities and had a pronounced impact on photosynthesis, the dicarboxylic acid metabolism, ribosomal metabolism, biosynthesis of secondary metabolites, glutathione metabolism and protein transport processing, which suggests that NO could accelerate plants metabolism, increase energy supply of metabolic activity, enhanced the capacity of ROS-scavenging, maintained the cell integrity and homeostasis equilibrium, thsu enhancing the ability of anti-metal in Cu-treated periwinkle seedlings.