To investigate whether Tic20 can indeed form an ion channel, Tic20-proteoliposomes were subjected to swelling assays . Changes in the size of liposomes in the presence of high salt concentrations, as revealed by changes in the optical density, can be used to detect the presence of a poreforming protein. After addition of 300 mM KCl to liposomes and Tic20-proteoliposomes, their optical densities dropped initially, due to shrinkage caused by the increased salt concentration. However, the optical density of protein-free liposomes remained at this low level, showing no change in their size; whereas in the case of Tic20-proteoliposomes the optical density increased constantly with time. The increase in optical density strongly supports the presence of a channel in Tic20-proteoliposomes that is permeable for ions, thereby creating an equilibrium between the inner compartment of the proteoliposomes and the surrounding buffer. To exclude the possible effects of contaminating channel-forming proteins derived from the bacterial membrane and a protein inserted into the liposomes , a further negative control was set up: Tic110 containing only the first three transmembrane helices was purified similarly to Tic20 and reconstituted into liposomes. We chose this construct, since NtTic110 inserts into the membrane during in vitro protein import experiments. Furthermore, as the full length and N-terminally truncated Tic110 possess very similar channel activities, it is unlikely that the N-terminal part alone forms a channel. The insertion of NtTic110 into liposomes was confirmed by incubation under different buffer conditions followed by flotation experiments, similarly to Tic20 . However,hydroponic dutch buckets these NtTic110-proteoliposomes behaved similarly to the empty liposomes during swelling assays: after addition of salt, the optical density decreased, and except for a small initial increase, it remained at a constant level.
This makes it unlikely that a contamination from E. coli or simply the insertion of a protein into the liposomes caused the observed effect in the optical density of Tic20- proteoliposomes. To further characterize the channel activity of Tic20, electrophysiological measurements were performed. After the fusion of Tic20-proteoliposomes with a lipid bilayer, ion channel activity was observed . The total conductance under symmetrical buffer conditions , 250 mM KCl was dependent on the direction of the applied potential: 1260 pS and 1010 pS under negative and positive voltage values, respectively. The channel was mostly in the completely open state, however, individual single gating events were also frequently observed, varying in a broad range between 25 pS to 600 pS. All detected gating events were depicted in two histograms.Two conductance classes were defined both at negative and positive voltage values with thresholds of 220 pS and 180 pS, respectively.The observed pore seems to be asymmetric, since higher conductance classes notably differ under positive and negative voltages. This is probably due to interactions of the permeating ions with the channel, which presumably exhibits an asymmetric potential profile along the pore. Since small and large opening events were simultaneously observed in all experiments, it is very unlikely that they belong to two different pores. The selectivity of Tic20 was investigated under asymmetric salt conditions , 250/20 mM KCl. Similarly to the conductance values, the channel is intrinsically rectifying ,supporting asymmetric channel properties. The obtained reverse potential is 37.0 ± 1.4 mV . According to the Goldman-Hodgkin-Katz approach, this corresponds to a selectivity of 6.5:1 for K+ :Cl- -ions, thus indicating cation selectivity similar to Tic110.
To determine the channel’s orientation within the bilayer, two side-specific characteristics were taken into account: the highest total conductance under symmetrical buffer conditions was measured under negative voltage values, and the channel rectifies in the same direction under asymmetrical buffer conditions . Therefore, it seems that the protein is randomly inserted into the bilayer. The pore size was roughly estimated according to Hille et al.. Considering the highest conductance class , a channel length of 1-5 nm and a resistivity of 247.5 Ω cm for a solution containing 250 mM KCl, taking into account that the conductivity of the electrolyte solution within the pore is ~5 times lower than in the bulk solution, the pore size was estimated to vary between 7.8-14.1 Å. This is in good agreement with the size of protein translocation channels such as Toc75 in the outer envelope membrane and Tic110 in the IE. Thus, the size of the Tic20 pore would be sufficient for the translocation of precursor proteins through the membrane. NtTic110, as a negative control, did not show any channel activity during electrophysiological measurements, indicating that the measured channel is not the result of a possible bacterial contamination . Considering our data presented here and those published in previous studies, we can conclude that the Tic translocon consists of distinct translocation channels: On the one hand, Tic110 forms the main translocation pore and therefore facilitates import of most of the chloroplast-targeted preproteins; on the other hand, Tic20 might facilitate the translocation of a subset of proteins. This scenario would match the one found in the inner mitochondrial membrane, where specific translocases exist for defined groups of precursor proteins: the import pathway of mitochondrial carrier proteins being clearly separated from that of matrix targeted preproteins. The situation in chloroplasts does not seem as clear-cut, but an analogous separation determined by the final destination and/or intrinsic properties of translocated proteins is feasible. The severe phenotype of attic20-I mutants prompts us to hypothesize that Tic20 might be specifically required for the translocation of some essential proteins. According to cross-linking results, Tic20 is connected to Toc translocon components. Therefore, after entering the intermembrane space via the Toc complex, some preproteins might be transported through the IE via Tic20.
On the contrary, Kikuchi et al. presented that Tic20 migrates on BN-PAGE at the same molecular weight as the imported precursor of the small subunit of Rubisco and that tic20-I mutants display a reduced rate of the artificial precursor protein RbcS-nt: GFP. The authors interpreted these results in a way that Tic20 might function at an intermediate step between the Toc translocon and the channel of Tic110. However,bato bucket being a substantial part of the general import pathway seems unlikely due to the very low abundance of Tic20. It is feasible to speculate that such abundant proteins as pSSU, which are imported at a very high rate, may interact incidentally with nearby proteins or indifferently use all available import channels. To clarify this question, substrate proteins and interaction partners of Tic20 should be a matter of further investigation. Additionally, a very recent study suggested AtTic20-IV as an import channel working side by side with AtTic20-I. However, detailed characterization of the protein and experimental evidence for channel activity are still missing.Cerium oxide nanoparticles are widely used in applications such as catalyst automotive industry, glass mirrors, plate glass, and ophthalmic lenses . These NPs are among the 13 engineered nanomaterials in the list of priority for immediate testing by the Organization for Economic Cooperation and Development . However, the environmental release of CeO2 NPs from factories or applications, and their behavior and effects in the environment are not well known yet . Previous studies have shown that CeO2 NPs are stable in soil at pH values of 7 to 9 . This suggests CeO2 NPs will remain in soil for a long time. In addition, reports from recent investigations have shown a wide variety of plant responses after exposure toCeO2 NPs. For instance, Schwabe found that CeO2 NP treatments did not reduced the growth in pumpkin and wheat. However, Ma et al. reported that, at 2000 mg/L, nano-CeO2 reduced root elongation in lettuce . Van Hoecke et al. found that CeO2 NPs, at concentrations as low as 2.6 and 5.4 mg/L, produced chronic toxicity to the unicellular alga Pseudokirchneriella subcapitata. Previous results from our research group have shown that CeO2 NPs at 2000 mg/L reduced corn and tomato germination by 30% and cucumber germination by 20% . In a more recent study, we demonstrated that CeO2 NPs are taken up and stored without change in maize roots . This previous study also revealed that the uptake of CeO2 NPs by corn plants was affected by soil organic matter content and alginate surface coating . Alginates are naturally occurring polysaccharides that have been used to stabilize NPs for several applications . This suggests that excess of alginate can be released into the environment together with NPs, with unknown consequences for edible plants. Thus, more studies are needed to better understand the impact of CeO2 NPs in plants, in environments where excess alginates could be present. On the other hand, studies have shown that carbon-based nanoparticles such as single walled carbon nanotubes triggered reactive oxygen species generation in Arabidopsis and rice .
In addition, multi-wall carbon nanotubes have been found to induce gene expression of heat shock protein 90 in tomato leaves and roots . However, there are no reports on the effect of CeO2 NPs on heat shock protein expression in plants. A few studies have described the physiological impacts of rare earth elements in plants. For example, at concentration higher than 89 µmol/L, cerium affected the foliar chlorophyll content, nitrate reductase activity, shoot root length and relative yield in cowpea plants . The authors suggested the effects could be produced by the substitution of Mg2+ by Ce in chlorophyll synthesis. It has also been suggested that, due to their similar chemical characteristics, Eu, a REE, may compete with Ca for organic ligands . These studies suggest that REE elements can have serious impacts on the uptake of nutritional elements in food crops. However, to the authors’ knowledge the impact of REE NPs on the uptake of nutritional elements by plants has yet to be reported. The purposes of this work were to determine the effects of alginate on: the transport of Ce within corn plants treated with CeO2 NPs, the uptake and transport of micro and macro nutrients, the chlorophyll content, and the expression of stress related heat shock protein 70. Maize was selected for this study because it is a crop widely cultivated throughout the world for direct and indirect consumption. In addition, 40% of the corn world’s harvest is produced in the United States . In this study, corn plants were grown in soil spiked with CeO2 NPs with various alginate concentrations for one month. After harvest, the concentration of Ce and many nutrient elements were determined by ICP-OES in the root and shoots tissues. This suggests that in an eventual release of CeO2 NPs, the higher risk of food contamination would occur in organic matter enriched soil. The mechanism involved in the increase of Ce uptake and translocation by alginate is still unknown. However, our previous work showed that alginate surface coating increased the Ce translocation to shoots in corn plants grown in a soil with low organic matter content and treated with 400 mg/kg CeO2 NPs. Sodium alginate has been associated with seed germination, shoot elongation, root growth, and flower production, among others in Foeniculum vulgare Mill . However, the mechanisms of these effects are still unknown. The presence of CeO2 NPs with/without alginate did not alter the uptake of macro-nutrients Mg, K, Ca, S, and P in one-month old corn roots. However, the uptake of Al and the micro-nutrients Fe, Mn, and Zn was increased . Compared to control , the concentrations of Fe and Al were significantly higher in all NP treatments. For Al, the difference was significant at p ≤ 0.023, but for Fe, the significance was only at p ≤ 0.09. The accumulation of both Fe and Al in roots was similar in all treatments. Moreover, compared to NPs alone and NPs-low alginate, the concentrations of both Fe an Al were significantly higher at medium and high alginate concentrations. It is very likely that the CeO2 NPs were bound with Fe and Al oxides, which are widespread soil colloids. Previous results showed that Fe and Al are co-released from the soil column with ZnO NPs . Manganese accumulation pattern was different. The addition of CeO2 NPs without alginate increased Mn accumulation in roots by 34% compared to control ; but NPs-low alginate and NPs-medium alginate treatments increased the accumulation of Mn by 92% and 90% respect to NPs without alginate and 158% and 155% respect to control. These differences were significant at p ≤ 0.005.