MafG-like contribute to copper and cadmium induced antioxidant response by regulating antioxidant enzyme in Procambarus clarkii.

Avian musculoaponeurotic fibrosarcoma (Maf) proteins play an important role in Nrf2/Keap1 signaling pathway, which mainly resist the oxidant stress. The members of sMaf have a high homology basic leucine zipper (bZIP) and lack trans activation domain, and could interact with other transcriptional regulatory factors as a molecular chaperone. In this study, a full-length MafG-like gene was cloned from Procambarus Clarkii, designated as PcMafG-like, which consisted of an ORF length of 246 bp encoding 82 amino acids, a 5' untranslated region (UTR) of 483 bp, and a 3' UTR of 111 bp. The domain of PcMafG-like had a bZIP-Maf domain that binds to DNA. The cDNA sequence of PcMafG-like was 99 % similar to that of Penaeus vannamei. The mRNA of PcMafG-like was expressed in all tested tissues, and the highest expression was in muscle tissue. Under stimulation of Cu2+ and Cd2+, PcMafG-like was significantly up-regulated in hepatopancreas and gill, and the same result was testified by situ hybridization. The representative antioxidant genes, CAT, GPx and CZ-SOD, were significantly induced by Cu2+; CAT and GPx was induced by Cd2+. PcMafG-dsRNA significantly inhibited the expression of these up-regulated genes, but also inhibited the expression of other detected genes CZ-SOD, GST-θ and GST-1like. The antioxidant effect of PcMafG-like was further verified by oxidative stress markers (T-SOD, CuZnSOD, GPx, CAT, GSH and MDA) kits. Cu2+ and Cd2+ could induce the contents of these oxidative stress markers (MDA, GSH, CZ-SOD, CAT in Cu2+/Cd2+ treated group, and GSH-Px in Cd2+ group), while interference of PcMafG-like significantly inhibited the up-regulation. Furthermore, hematoxylin-eosin staining experiments showed that the degree of pathological damage was dose-dependent and time-dependent, and the pathological damage was more serious after dsRNA interfered with PcMafG-like. In addition, subcellular localization showed that PcMafG-like gene existed in nucleus. The recombinant protein PcMafG-like was expressed and purified in prokaryotic expression. The affinity analysis of promoter by agarose gel electrophoresis suggested that PcMafG-like could bind with CAT promoter in vitro. This indicated that PcMafG-like could activate antioxidant genes.

Subcellular localization of copper in tolerant and non-tolerant plant.

The ability of Elsholtzia splendens Naki (E. splendens) to accumulate copper appears to be governed by its high degree of coppertolerance. However, the tolerance mechanism on the physiological basis is unknown. Using transmission electron microscope (TEM) and energy dispersive analysis of X-rays (EDX), the likely location of copper within the cells of the tolerant and non-tolerant was determined. Here the role of vacuolar and cell wall compartmentalization in this copper tolerant plant were investigated. A direct comparison of copper locations of E. splendens and the non-tolerant Astragalus sinicus L. (A. sinicus) showed that the majority of copper in the tolerant was localized primarily in the vacuolar, cell wall, on the plasmamembrane, beside lipid grains induced by copper pollution, in the chloroplasts and amyloids; but in the non-tolerant, copper precipitates only be observed on the plasmamembrane, in the chloroplasts and cytoplasm under copper exposure conditions that were toxic to both species. This revealed that the tolerant accumulates more copper in the vacuole and cell wall than the non-tolerant, where was regarded as the storage compartment of tolerant plant or hyperaccumulator for heavy metals.

[Change of superficial shape and cell microstructure of milk-vetch (Astragalus L.) root under simulated Cu pollution in a red soil].

This study carried out experiments to investigate changes of shape, inter-structures and cell microstructures of milk-vetch root under simulated Cu stress at non- or contaminated levels in a red soil using observation and bio-microscopic technique. It resulted that when Cu concentration ranged from 0 to 40 mg.kg-1 soil, the milk-vetch grew well and it had a whole root which worked normally. When Cu concentration reached 50 mg.kg-1 soil, the growth of milk-vetch began to get influence with decline of biomass, the taproot crooked and was less branched, root became short and hazel and had fewer shorter hairs, tubby appeared, epidermis began to shrink and cell wall cockled slightly and unevenly, the boundary between plasmalemma and organelle blurred as well. When treatment concentration reaching to 200 mg.kg-1 soil, milk-vetch roots became rotted and black, the cell wall broke and cytoplasm shrank so severely that plasmolysis happened and the plant died. So the critical Cu concentration in experimented soil was 50 mg.kg-1 soil, and the resistance of milk-vetch root to Cu contamination buildup with the growth of aboveground part, and cell wall was the main part to Cu tolerance.