Cadmium-induced Changes In Oxidative Metabolism And Calcium Messenger Of Plant And Their Molecular Mechanisms

The question as to how cadmium (Cd) acts at the cellular and molecular level and how plants may defend themselves against this pollutant has been paid increasing attention. To investigate how Cd induces common plant defense pathways can provide basis for improving Cd tolerance of plants, and is also helpful for understanding of environmental toxicology for animal and human as Cd²⁺ can also bond to N- and S- of biological big molecules and then interfere oxidative metabolism and induce changes in calcium messenger.To gain better insight into the biological mechanisms of cadmium-induced changes in oxidative metabolism and calcium messenger in plants, maize (Zea mays) seedlings were exposed to different Cd²⁺ concentration (0, 5, 20 and 100μmol/L Cd) in hydroponics, the temporal sequence(12﹑24﹑48﹑96 and 168 h)of physiological reactions, including (1) oxidative metabolism such as hydrogen peroxide (H₂O₂) production, activities of superoxide dismutases (SOD), catalases (CAT), ascorbate peroxidases (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR), and concentrations of ascorbate and glutathione, and gene expression of SOD, CAT, APX, DHAR and GR; (2) involvement of calcium messenger such as changes in CaM content and Ca²⁺-ATPase activities, and their related gene expression, and (3) subcelluar distribution of cadmium in maize was characterized. Biochemical procedure were used to determine the amount/ activities of the related enzyme, protein or products in oxidative metabolism and calcium messenger, the related genes were isolated from maize leaves using rapid amplication of cDNA ends (RACE), and Northern hybridization was applied to identify the stage-specific expression. The subcellular distribution of Cd in maize roots and leaves was probed with Transmission Electron Microscope --Energy-dispersive X-ray Microanalysis (TEM-EDXMA).The main findings obtained are as follows:1.Biomass of maize leaves and roots declined with increasing Cd supply level, the growth inhibition was higher with prolonged Cd exposure time. It was found that the biomass of maize leaves and roots after incubating for 168h in the treatment of 100μmol/L Cd was 52.8% and 33.3% lower than that without Cd addition, respectively. Cd accumulation in maize plants exposed to Cd increased with increasing Cd supply level and prolonged Cd exposure time. It was generally observed that the highest Cd peak in root arose in cell wall, and that in leaf followed a tendency of vacuole > cell wall > cytoplasm,indicating that Cd toxicity in plants was alleviated by sequestration of Cd into vacuole and apoplast (cell wall) under Cd stress.2.O2.- generation rate,H₂O₂ and Malondialdehyde (MDA) content in leaves and roots increased rapidly with increasing Cd concentrations and prolonged Cd exposure time. Both SOD activity and Cu/Zn-SOD gene expression were induced by Cd and increased at first, then declined considerably in the treatment of 5 and 20μmol/L Cd after incubating for 48h, however, those at 100μmol/L Cd remained a higher level after incubating for 168h. Therefore, the expression of Cu/Zn-SOD gene was regulated by Cd at transcription level as the gene expression pattern is consistent with the change of this enzyme activity.CAT activity in maize leaves was induced by Cd exposure and increased at first in the treatment of 5 and 20μmol/L Cd, then depressed gradually after incubating for 24h, while that with Cd supply level of 100μmol/L Cd was even lower than that of control throughout the entire incubation period. CAT gene expression in maize leaves was induced by Cd in the treatment of 20 or 100μmol/L Cd, then declined slightly after incubating for 24h, but remained a markedly higher level during entire incubation in comparison with the control, indicating that CAT gene expression might be regulated by post–transcriptional or post–translational modification in maize leaves under Cd stress.APX activity in maize leaves was enhanced by Cd exposure, and was 24%, 67% and 50% higher than control after incubating for 12h in the treatment of 5, 20 and 100μmol/L Cd, respectively, then decreased gradually, even lower than that of control after incubating for 168h. APX gene expression in maize leaves was induced by Cd in the treatment of 5 or 20μmol/L Cd, and remained a higher level after incubating for 168h, while that in the treatment of 100μmol/L Cd was inhabited, and lower than that with 20μmol/L Cd during entire incubation, suggesting that APX gene expression might be regulated by post–transcriptional or post–translational modification.3.The Ascorbate- Glutathione cycle (AsA-GSH cycle) in leaves of maize could not be induced obviously by Cd exposure at low Cd supply level. The total ascorbate, reduced ascorbate (AsA) and dehydroascorbate (DAsA) contents in leaves of maize incubated at a lower Cd supply level remained a relatively stable level with prolonged Cd exposure time throughout the entire incubation period. Both DHAR activity and gene expression in the each treatment of Cd addition was generally found to be lower, and similar to that without Cd addition, showing that DHAR gene transcription could not be induced with a lower Cd supply level. It was observed that GR activity and total ascorbate content in leaves were enhanced significantly by Cd exposure with 100μmol/L Cd after incubating for 168h, indicating that AsA-GSH cycle could be activated by a prolonged Cd exposure time at a higher Cd supply level.4.The sequence of Ca²⁺-ATPase activities in cell membrane systems was plasma membrane >tonoplast membrane,endoplasmic reticulum membrane> mitochondrial membrane.Ca²⁺-ATPase activities in cell membrane systems were enhanced by Cd at first, then began to decline gradually in plasma, tonoplast and endoplasmic reticulum membrane after incubating for 48 h, and in mitochondrial membrane after incubating for 24 h. The profile of plasma membrane Ca²⁺-ATPase gene expression was discovered to be in accordance with the changes of this enzyme activity in maize leaves. Both CaM content and gene expression in leaves was up-regulated with increasing Cd concentrations and prolonged Cd exposure time in comparison with that of control, the changes in CaM content was found to be consistent with the temporal expression profile. Therefore, gene expression of both plasma membrane Ca²⁺ -ATPase and CaM were enhanced by Cd at transcription level.Based on above, antioxidant enzyme activities in oxidative metabolism in maize leaves can be induced by cadmium, the expression of Cu/Zn-SOD gene was enhanced by Cd at transcription level, while those of CAT and APX gene were up-regulated at the translational or post– translational level. It seems that the AsA-GSH cycle in leaves of maize could not be activated obviously by low Cd supply level, while enhanced by a prolonged Cd exposure time with a higher Cd supply level. For calcium messenger, both Ca²⁺ -ATP ase and CaM gene expression could be up-regulated by Cd exposure at transcription level, and accordingly enhanced Ca²⁺ -ATP ase activities and CaM contents in leaves of maize.