Cell cycle arrest mediated by Cd-induced DNA damage in Arabidopsis root tips

Accumulating evidence demonstrates that the aberrant expression of cell cycle regulation and DNA repair genes can result in abnormal cell proliferation and genomic instability in eukaryotic cells under different stresses. Herein, Arabidopsis thaliana (Arabidopsis) seedlings were grown hydroponically on 0.5 × MS media containing cadmium (Cd) at 0–2.5 mg L−1 for 5 d of treatment. Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that expression of DNA damage repair and cell cycle regulation genes, including BRCA1, MRE11, WEE1, CDKA;1 and PCNA1, showed an inverted U-shaped dose-response. In contrast, notably reduced expression was observed for G1-to-S transition-related genes, Histone H4, E2Fa and PCNA2; DSB end processing, GR1; G2-to-M transition-related gene, CYCB1;1; and DNA mismatch repair, MSH2, MSH6 and MLH1 genes in root tips exposed to 0.125–2.5 mg/L Cd for 5 d. Flow cytometry (FCM) analysis revealed significant increases of cells with a 2C nuclear content and with a 4C and 8C nuclear content under Cd stresses of 0.125 and 1–2.5 mg L−1, respectively. Our results suggest that 0.125 mg L−1 Cd-induced DNA damage induced the marked G1/S arrest, leading to accelerated growth in root tips, while 1.0–2.5 mg L−1 Cd-induced DNA damage caused a notable G2/M arrest in root tips, leading to reduced growth in root tips. This may be a protective mechanism that prevents cells with damaged DNA from dividing under Cd stress

Cadmium-induced genomic instability in Arabidopsis: molecular toxicological biomarkers for early diagnosis of cadmium stress

Microsatellite instability (MSI) analysis, random-amplified polymorphic DNA (RAPD), and methylation-sensitive arbitrarily primed PCR (MSAP-PCR) are methods to evaluate the toxicity of environmental pollutants in stress-treated plants and human cancer cells. Here, we evaluate these techniques to screen for genetic and epigenetic alterations of Arabidopsis plantlets exposed to 0–5.0 mg L−1 cadmium (Cd) for 15 d. There was a substantial increase in RAPD polymorphism of 24.5, and in genomic methylation polymorphism of 30.5–34.5 at CpG and of 14.5–20 at CHG sites under Cd stress of 5.0 mg L−1 by RAPD and of 0.25–5.0 mg L−1 by MSAP-PCR, respectively. However, only a tiny increase of 1.5 loci by RAPD occurred under Cd stress of 4.0 mg L−1, and an additional high dose (8.0 mg L−1) resulted in one repeat by MSI analysis. MSAP-PCR detected the most significant epigenetic modifications in plantlets exposed to Cd stress, and the patterns of hypermethylation and polymorphisms were consistent with inverted U-shaped dose responses. The presence of genomic methylation polymorphism in Cd-treated seedlings, prior to the onset of RAPD polymorphism, MSI and obvious growth effects, suggests that these altered DNA methylation loci are the most sensitive biomarkers for early diagnosis and risk assessment of genotoxic effects of Cd pollution in ecotoxicology

Roles of MSH2 and MSH6 in cadmium-induced G2/M checkpoint arrest in Arabidopsis roots

DNA mismatch repair (MMR) proteins have been implicated in sensing and correcting DNA damage, and in governing cell cycle progression in the presence of structurally anomalous nucleotide lesions induced by different stresses in mammalian cells. Here, Arabidopsis seedlings were grown hydroponically on 0.5 × MS media containing cadmium (Cd) at 0–4.0 mg L−1 for 5 d. Flow cytometry results indicated that Cd stress induced a G2/M cell cycle arrest both in MLH1-, MSH2-, MSH6-deficient, and in WT roots, associated with marked changes of G2/M regulatory genes, including ATM, ATR, SOG1, BRCA1, WEE1, CYCD4; 1, MAD2, CDKA;1, CYCB1; 2 and CYCB1; 1. However, the Cd-induced G2/M phase arrest was markedly diminished in the MSH2- and MSH6-deficient roots, while a lack of MLH1 had no effect on Cd-induced G2 phase arrest relative to that in the wild type roots under the corresponding Cd stress. Expression of the above G2/M regulatory genes was altered in MLH1, MSH2 and MSH6-deficient roots in response to Cd treatment. Furthermore, Cd elicited endoreplication in MSH2- and MSH6-deficient roots, but not in MLH1-deficient Arabidopsis roots. Results suggest that MSH2 and MSH6 may act as direct sensors of Cd-mediated DNA damage. Taken together, we conclude that MSH2 and MSH6, but not MLH1, components of the MMR system are involved in the G2 phase arrest and endoreplication induced by Cd stress in Arabidopsis roots

The exposure of gadolinium at environmental relevant levels induced genotoxic effects in Arabidopsis thaliana (L.)

Rare Earth Elements (REEs) are increasingly being used in agriculture and are also used to produce high end technological devices, thereby increasing their anthropogenic presence in the environment. However, the ecotoxicological mechanism of REEs on organisms is not fully understood. In this study, the effects of gadolinium (Gd) addition on Arabidopsis thaliana (L.) were investigated at both physiological and molecular levels. Four treatments (0, 10, 50 and 200 μmol·L−1 Gd) were used in the exposure tests. Biomass, root length and chlorophyll content in shoots/roots were measured to investigate the plant’s physiological response to Gd stress. Random amplified polymorphic (RAPD)-Polymerase Chain Reaction (PCR) and methylation sensitive arbitrarily primed (MSAP)-PCR were used to investigate changes in genetic variation and DNA methylation of A. thaliana when exposed to Gd. At the physiological level, it was found that low concentration of Gd (10 μmol·L−1) could significantly increase the plant biomass and root length, while the growth of A. thaliana was significantly inhibited when exposed to 200 μmol·L−1 of Gd, yet the total soluble protein content in aerial plant parts increased significantly by 24.2% when compared to the control group. Among the 12 primers considered in the RAPD assessment, at the molecular level, only four primers revealed different patterns in their genomic DNA. Compared to the control group, the treatment with 50 μmol·L−1 of Gd was associated with lower polymorphism, while the treatment with 200 μmol·L−1 of Gd was associated with higher polymorphism. The polymorphism frequencies for the 50 μmol·L−1 of Gd and the 200 μmol·L−1 of Gd were 4.67% and 20.33%, respectively. The MSAP analysis revealed that the demethylation (D) type of Arabidopsis genomic DNA increased significantly under 10 and 50 μmol·L−1 of Gd, while the methylation (M) type was also significantly increased under 200 μmol·L−1 of Gd. Generally, the total methylation polymorphism (D+M) increased with an increase of Gd concentration. It was found that high concentrations of Gd appeared to cause DNA damage, but low concentrations of Gd (as low as 10 μmol·L−1) were associated with DNA methylation change. Further, it was verified by Real time Reverse Transcription PCR (RT-PCR) on the bands detected by the MSAP analysis, that the genes relative to processes including cell cycle, oxidative stress and apoptosis, appeared to be regulated by methylation under Gd stress. These findings reveal new insight regarding ecotoxicity mechanisms of REEs on plants

Long-distance transport of cadmium from roots to leaves of Solanum melongena

In this study, the characteristics of cadmium (Cd) uptake by roots and translocation from roots to leaves of two eggplant species (Solanum melongena and Solanum torvum) under relatively low Cd concentrations were investigated using stable 108Cd isotope through a number of hydroponic experiments. The uptake and translocation of 108Cd was compared with those of 70Zn and 15N. The results showed more 108Cd was loaded to the vascular channels and translocated upward to the leaves in S. melongena than in S. torvum, while the 108Cd concentrations were significantly lower in the roots of S. melongena than in S. torvum. When the phloem and xylem were wounded by grafting treatments, the foliar 108Cd concentrations were decreased by more than 66 % regardless of the rootstock species, whereas the uptake of 108Cd in the root was not inhibited by grafting. Similar grafting effects were observed for 70Zn. Hence, wounding phloem and xylem by grafting disturbed the upward transport of 108Cd and 70Zn to the eggplant leaves. Similarly, interruption of the phloem by the girdling treatment reduced the concentrations of 108Cd in the leaves of S. melongena by approximately 51 %, though the uptake of 108Cd by roots was not reduced by the interruption of phloem. In contrast, neither 70Zn concentrations nor stable N isotope ratio (δ15N) values in the roots and leaves of S. melongena were significantly influenced by the interruption of phloem. In conclusion, the phloem played a dominant role in the long-distance transport of Cd from the root to the leaf of S. melongena, whereas the xylem was the main channel for the translocation of Zn and N

The effect of concentrations and properties of phenanthrene, pyrene, and benzo(a)pyrene on desorption in contaminated soil aged for 1 year

Purpose: The choice and timing of microorganisms added to soils for bioremediation is affected by the dominant bioavailable contaminants in the soil. However, changes to the concentration of bioavailable PAHs in soil are not clear, especially when several PAHs coexist. This study investigated the effects of PAH concentration and chemical properties on desorption in meadow brown soil after a 1-year aging period, which could reflect changes of PAH bioavailability during bioremediation. Materials and methods: Based on the percentage of different molecular weights in a field investigation, high-level contaminated soil (HCS) and low-level contaminated soil (LCS) were prepared by adding phenanthrene (PHE), pyrene (PYR) and benzo(a)pyrene (BaP) to uncontaminated meadow brown soil. The concentrations of HCS and LCS were 250 mg kg-1 (PHE, PYR, and BaP: 100, 100, and 50 mg kg-1) and 50 mg kg-1 (PHE, PYR, and BaP: 20, 20, and 10 mg kg-1) respectively. The soils were aged for 1 year, after which desorption was induced by means of a XAD-2 adsorption technique over a 96-h period. Results and discussion: The range of the rapidly desorbing fraction (Frap) for PHE, PYR, and BaP in HCS and LCS was from 1. 9 to 27. 8 %. In HCS, desorption of PYR was most difficult, and the rate constant of very slow desorption (Kvs) of PYR was 8 orders of magnitude lower than that of BaP, which had similar very slow desorbing fractions (49. 8 and 50. 5 %, respectively). However, in LCS, desorption of PYR was the easiest; the Kvs of PYR was 8-10 orders of magnitude higher than those of PHE and BaP. In HCS, the time scale for release of 50 % of the PAHs was ranked as BaP & PYR & PHE, while in LCS this was BaP & PHE & PYR. Conclusions: The combined effect of PAH concentrations and properties should be taken into account during desorption. The desorption of PAH did not always decrease with increasing molecular weight, and the desorption of four-ring PAHs might be special. These results are useful for screening biodegrading microbes and determining when they should be added to soils based on the dominant contaminants present during different periods, thus improving the efficiency of soil bioremediation. © 2012 Springer-Verlag Berlin Heidelberg