| In this study, common aquatic plants such as Alternanthera philoxeroides and Sagittaria sagittifolia were taken as experiment materials, and the effects of heavy metal Cu2+on reactive oxygen species (ROS) level and polyamine metabolism in these plants were investigated. The alleviation effect of exogenous spermidine (Spd) on Cu2+toxicity in Alternanthera philoxeroides and the effect of Cu2+on ROS level and anti-oxidative system in Alternanthera philoxeroides callus were also involved. Moreover, the SSH library of Potamogeton crispus under Cu2+stress was structured. Genes expressed differently in the SSH library were analyzed. Concrete results as follow:(1) Effects of Cu2+on the ROS level and polyamine metabolism in Alternanthera philoxeroides leaves were investigated. Cu2+treatment induced a marked accumulation of Cu and enhanced contents of thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2) and the generation rate of O2·-. It also significantly increased putrescine (Put) level but lowered spermidine (Spd) and spermine (Spm) levels. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and polyamine oxidase (PAO) were all elevated with the increase of Cu+concentration. However, application of exogenous Spd effectively decreased H2O2content and the generation rate of O2·-, prevented Cu2+-induced lipid peroxidation and reduced Cu accumulation. Moreover, it declined level of endogenous Put and increased levels of Spd and Spm. Activities of ADC, ODC and PAO were all inhibited by exogenous Spd. The results indicated that application of exogenous Spd could enhance the tolerance of A. philoxeroides to Cu2+stress by reducing ROS level and balancing polyamine metabolism.(2) Effects of Cu2+on the roots of Sagittaria sagittifolia were studied after treatment at five concentration levels. The accumulation of Cu, the generation rate of O2·-,the contents of TBARS and polyamine, as well as the activities of ADC and PAO in the roots were measured and analyzed. The goal of the present study was investigating the effects of Cu contamination on the above-mentioned biochemical and physiological parameters in order to explore possible prevention strategies against heavy metal stress. It was observed that endogenous Cu content increased in roots of S. sagittifolia in a concentration-dependent manner, along with an increased production of O2·-. TBARS content increased progressively up to5μmol·L-1Cu2+. A constant increase in ADC activity was also observed. The results indicated that lower Cu2+concentrations (2.5and5μmol·L-1, respectively) had greater enhancing effect on the contents of free Put and perchloric acid-soluble conjugated (PS-conjugated) Put, while Cu2+treatments at different concentration levels had similar enhancing effect on the content of perchloric acid-insoluble bound (PIS-bound) Put. In total, Put content in each Cu-treated group was higher than that in the control group. PAO activity was inhibited up to10μmol·L-1Cu2+but enhanced at higher Cu2+concentrations (20and40μmol·L-1). This explained the initial rise and subsequent decline of the contents of all forms of spermine (Spm), free and PS-conjugated spermidine (Spd). However, with the increase of Cu2+concentration, total Spm content increased gradually while total Spd content decreased. Our results suggest that Cu2+is phytotoxic to the roots of S. sagittifolia at high concentrations, and that the increased Spm level is not sufficient to resist Cu-induced oxidative damages.(3) Callus of Alternanthera philoxeroides was subjected to different concentrations of Cu2+and contents of soluble protein and TBARS, activities of superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (G-POD), levels of ROS (such as O2-and H2O2) were determined. Treatment with0.05mmol·L-1Cu2+enhanced the content of soluble protein and reduced levels of O2--and H2O2. As Cu2+concentration exceeded0.1mmol-L"1, the content of soluble protein decreased greatly and the amounts of two polypeptides (apparent molecular weights45and39kDa) became visible in SDS-PAGE; SOD activity enhanced markedly owing to the accumulation of O2-" while POD and CAT activities increased alternately in response to increased H2O2content. Because of congenerous function of POD and CAT, H2O2content dropped down to a low level at last. An interesting result was that TBARS content decreased with the increasing concentration of Cu2+. The results of this study indicated that antioxidant enzymes (SOD, POD and CAT) protected A. philoxeroides callus against copper stress efficiently, especially for SOD.(4) In order to investigate genes which were expressed differently in Potamogeton crispus under Cu+stress, bidirection SSH libraries of P. crispus with different treatments were constructed by SSH technique. In forward SSH library, P. crispus treated with Cu+was used as tester and that treated without Cu2+was used as driver. In backward SSH library, the tester and driver were interchanged. The total RNAs from the two groups were isolated respectively, then the single strand and double strand cDNA (ds cDNA) were synthesized. After Rsa I digested the ds cDNA, ds cDNA of the tester were added to adaptor1and adaptor2R. The hybridization products were obtained through two times hybridization and two times PCR. The PCR products were cloned into pED-T vectors which were transformed into DH5a. Massive positive clones in the SSH library were sequenced, and made GO analysis through Gene Ontology database and pathway analysis through KEGG database. The analytical results indicated that most of the genes which expressed differently in the SSH library were associated with some metabolism pathways and involved in enzymes in the pathway.
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