Effects Of Chloroplast-Targeted CadR On Cadmium Tolerance Of Transgenic Arabidopsis

The unprecedented increase in the environmental level of heavy metals has become a global concern. Heavy metals are released into the environment through geogenic and anthropogenic activities, such as rocky outcropping, volcanic eruption, coal-burning, urban-industrial emissions, automobile exhausts and sewage disposals. Cadmium(Cd) stands 7th out of the 20 toxins and is widely distributed in the environment. As Cd cannot be easily degraded and its relative mobility, its discharge into marine and terrestrial ecosystems as well as accumulation in food chain, so it has posed a serious threat to mankind. Exposure to high levels of Cd has been found to be carcinogenic, mutagenic and teratogenic for a large number of living organisms.Recently, it has been reported that Cd can induce pro-inflammatory effects in human lung adenocarcinoma epithelial cells. In plants, Cd accumulation interferes with photosynthesis, water homeostasis, gas-exchange, nutrient uptake, membrane function and antioxidant system, thus resulting in visible injury symptoms such as chlorosis, growth inhibition, browning of root tips, and death.The photosynthetic apparatus is sensitive to many heavy metals including Cd. Under Cd stress, chloroplasts suffer severe alterations of their ultrastructure, which are associated with profound molecular and metabolic damages. For example, Cd has negative effects on chlorophyll biosynthesis, electron transport and enzymes of photosynthetic carbon metabolism. Given highly oxidizing metabolic nature and the presence of electron transport chains(ETCs) in chloroplasts, formation of excess reactive oxygen species(ROS) is inevitable with increasing concentrations of Cd. Inhibition of photoactivation in photosystem ?(PS?), decrease of photosynthetic net rate and suppression of photorespiration processes have been attributed to disruptive action of Cd in algea. Many efforts are underway to engineer overexpression of metal-binding proteins in chloroplasts to alleviate metal toxicity and to subsequently improve metal resistance.Prokaryotes typically carry a panel of metalloregulatory proteins that directly bind metals and collectively manage metal ion homeostasis and resistance. These individual metalloregulatory proteins selectively respond to one or a small overlapping subset of metal ions. A key class of prokaryotic metalloregulatory proteins is the MerR family, of which a Cd sensing protein, CadR, controls the expression of genes responsible for Cd detoxification in bacteria. cadR encodes a 147-amino-acid protein and its expression is exclusively induced by Cd. Similar to other MerR-family members, CadR contains N-terminal helix-turn-helix motifs for DNA binding and three conserved cysteine residues(Cys77, Cys112 and Cys119) for Cd binding. In addition, CadR has an unusual histidine-rich C-terminal that contributes to its high specificity towards Cd.The aim of this research is to demonstrate the use of Cd-specific cadR gene from the rhizobacterium Pseudomonas putida for improvement of Cd accumulation and Cd resistance in plants. The model plant, Arabidopsis thaliana, is used to harbor gene expression cassette that expresses CadR in cytoplasm or targets CadR into chloroplasts. Thus when Cd is present, Cd can be chelated by CadR in cytoplasm or in chloroplasts. The effects of cytoplasmic and chloroplastic CadR on Cd accumulation and resistance of the transgenic plants were compared. We show that the plants with chloroplastic CadR exhibited strong ability to accumulate Cd and to resist Cd. Importantly, the resistance of the genetically modified plants was highly specific to Cd(?), while these plants did not respond to other valence II cations.The main results are as follows:(1) Generation of transgenic Arabidopsis plants expressing chloroplast-targeted CadRTo target CadR to plastids, we ligated a transit peptide(TP) of ArabidopsisRbc S(small subunit of theRubisco complex) to the upstream of CadR. In addition, we used shoot-specific chlorophyll a/b-binding protein 2 gene(CAB2) promoter to specifically express TP-CadR in leaf chloroplasts.The subcellular localization of CadR-GFP/TP-CadR-GFP were analyzed by confocal laser scanning microscopy(CLSM). Green fluorescence was found in both shoots and roots of the p35S::cadR-GFP and p35S::TP-cadR-GFP plants, but only in shoots of the p CAB2::TP-cadR-GFP plants.(2) Expression of cadR enhances Cd toleranceUnder Cd exposure, roots of all transgenic plants were longer than the WT plants. For example, at 25 ?M Cd, the transformants showed an average 54% increase in root elongation compared with the WT plants. No significant differences in fresh weight between the transgenic lines and the WT plants were observed at 25, 50 and 100 ?M Cd except at 75 ?M Cd, where the transgenic lines showed a 27–57% increase relative to the WT plants.(3) Cd accumulation increased in cadR transgenic ArabidopsisIn response to short-term(2 d) exposure to Cd, the transgenic lines exhibited a significant increase(up to ~2-fold) in Cd content in roots, but maintained a similar level of Cd in leaves, compared with the WT plants. Upon exposure to Cd for a mid term(7 d), both the transgenic and the WT plants accumulated more Cd in leaves compared with those under short-term(2-d) exposure to Cd. However, there was no obvious differences in leaf Cd contents between the transgenic and the WT plants despite root Cd contents in the transgenic plants were much higher than that in the WT plants. Under long-term(16 d) Cd exposure, Cd contents in leaves and roots of all cadR transgenic plants were significantly higher than those of the WT plants. We then calculated total Cd accumulation(?g per plant) of shoot(including leaves, cauline leaves, stems and siliques) and root in the transgenic and WT plants under exposure to Cd. Total Cd contents in roots and shoots of all plants were increased with Cd exposure time. Under mid-term(7-d) and long-term(16-d) exposure to Cd, total Cd accumulation in shoots and roots of the transgenic plants were significantly higher(up to 3.5-fold increase in shoots and 5.1-fold increase in roots) than those of the WT plants.(4) Effects of Cd on mineral nutrient accumulation in the transgenic plantsA quantitative analysis of macronutrients(such as Ca, K, P, S, Na and Mg) and micronutrients(such as Mn, Zn, Cu and Fe) was performed in the WT plants and the transgenic lines under long-term(16 d) exposure to Cd. Mn contents in leaves and roots of the transgenic plants were significantly higher(up to 2.5-fold in leaves and 3.0-fold in roots) than those of the WT plants. Mn levels in cauline leaves and stems were 0.7–2.0-fold above levels of the WT plants. Other metal elements accumulated at a slightly lower level in roots of the transgenic plants than those of the WT plants.(5) Expression of CadR exerts protective effects on chlorophyll and carotenoid levelsExposure to 100 ?M Cd resulted in remarkable decrease of Chl b levels in all plants. However, a less Cd-mediated decrease in Chl b was observed in the cadR transgenic plants compared with the WT plants, leading to Chl b contents in the transgenic plants were averagely 1.6-fold(p35S::cadR), 4.1-fold(p35S::TP-cadR) or 3.9-fold(p CAB2::TP-cadR) higher than that in the WT plants. After 100 ?M Cd exposure, a dramatic drop in carotenoid levels of p35S::cadR plants was detected, while p35S::TP-cadR and p CAB2::TP-cadR plants remained similar levels of carotenoid to the WT plants.(6)Responses to other divalent metal stressGrowth phenotypes of the cadR transgenic plants were very similar to that of the WT plants in response to other divalent metals, such as Hg, Pb, Cu and Zn.