Physiological Responses to Cadmium, Nickel and their Interaction in the Seedlings of Two Maize (Zea mays L.) Cultivars

In the leaves of maize seedlings, cultivars Premia and Blitz, the relatively low 2 μmol/L concentration of cadmium (Cd), nickel (Ni), or both metals acting simultaneously (Cd +Ni) for 72 h, induced a significant metal accumulation, decrease in total K+ content, reduction of light-induced membrane electrical potential (EM) repolarisation in mesophyll cells and changes of ascorbic acid (AsA), dehydroascorbic acid (DHA) and glutathione (GSH) content. Shoot growth and the values of resting EM did not change significantly. Increased K+ leakage, from the leaves, and lipid peroxidation accompanied by increase of TBA-reactive substances (TBARS) were found only in cv. Blitz exposed to Cd + Ni. This indicates a capability of high leaf-cell anti-oxidant pool to ameliorate the toxic effects on plasma membrane of single ions in both cultivars, and of Cd + Ni only in cv. Premia. The decreased total content of K+ in leaves in all variants indicated repressing the K+ uptake and/or distribution to the shoots. Under anoxia, the magnitude of the repolarisation obtained after switching on the light was smaller in Cd-treated cultivar Premia than in the controls, and this also occurred in Ni- and Cd + Ni-treated cultivar Blitz.

Signaling mechanism by the Staphylococcus aureus two-component system LytSR: role of acetyl phosphate in bypassing the cell membrane electrical potential sensor LytS

The two-component system LytSR has been linked to the signal transduction of cell membrane electrical potential perturbation and is involved in the adaptation of Staphylococcus aureus to cationic antimicrobial peptides. It consists of a membrane-bound histidine kinase, LytS, which belongs to the family of multiple transmembrane-spanning domains receptors, and a response regulator, LytR, which belongs to the novel family of non-helix-turn-helix DNA-binding domain proteins. LytR regulates the expression of cidABC and lrgAB operons, the gene products of which are involved in programmed cell death and lysis. In vivo studies have demonstrated involvement of two overlapping regulatory networks in regulating the lrgAB operon, both depending on LytR. One regulatory network responds to glucose metabolism and the other responds to changes in the cell membrane potential. Herein, we show that LytS has autokinase activity and can catalyze a fast phosphotransfer reaction, with 50% of its phosphoryl group lost within 1 minute of incubation with LytR. LytS has also phosphatase activity. Notably, LytR undergoes phosphorylation by acetyl phosphate at a rate that is 2-fold faster than the phosphorylation by LytS. This observation is significant in lieu of the in vivo observations that regulation of the lrgAB operon is LytR-dependent in the presence of excess glucose in the medium. The latter condition does not lead to perturbation of the cell membrane potential but rather to the accumulation of acetate in the cell. Our study provides insights into the molecular basis for regulation of lrgAB in a LytR-dependent manner under conditions that do not involve sensing by LytS.

Impact of nickel on grapevine (Vitis vinifera L.) root plasma membrane, ROS generation, and cell viability

The present study investigated the impact of nickel (Ni2+) on trans-membrane electrical potential (EM) and permeability properties of plasma membrane (PM) in epidermal cells of adventitious grapevine roots. The relationship between disturbances of membrane functionality and the production of superoxide anion, hydrogen peroxide and cell viability after the exposure of roots to Ni2+ was also studied. Treatments with 0.1-5 mmol L-1 NiCl2 induced a concentration-dependent transient PM depolarization, which was recovered to the initial resting potential within 50-70 min in the presence of Ni2+. Longer (up to 24 h) exposure of roots to 1 mmol L-1 of Ni2+ hyperpolarized the EM by approximately 17 mV. Application of the highest 5 mmol L-1 concentration of Ni2+ during longer treatments (up to 48 h) resulted in the increase of membrane permeability; however the EM, cell viability, and superoxide content remained unaffected. The increase in the formation of hydrogen peroxide was time- and concentration- dependent and maximum production was recorded after 180 min of Ni2+ treatment. We can conclude that oxidative stress resulting from an imbalance in the generation and/ or removal of hydrogen peroxide in the root tissues of grapevine was the major cause of Ni2+ toxicity.

Signaling mechanism by the Staphylococcus aureus two-component system LytSR: role of acetyl phosphate in bypassing the cell membrane electrical potential sensor LytS

The two-component system LytSR has been linked to the signal transduction of cell membrane electrical potential perturbation and is involved in the adaptation of Staphylococcus aureus to cationic antimicrobial peptides. It consists of a membrane-bound histidine kinase, LytS, which belongs to the family of multiple transmembrane-spanning domains receptors, and a response regulator, LytR, which belongs to the novel family of non-helix-turn-helix DNA-binding domain proteins. LytR regulates the expression of cidABC and lrgAB operons, the gene products of which are involved in programmed cell death and lysis. In vivo studies have demonstrated involvement of two overlapping regulatory networks in regulating the lrgAB operon, both depending on LytR. One regulatory network responds to glucose metabolism and the other responds to changes in the cell membrane potential. Herein, we show that LytS has autokinase activity and can catalyze a fast phosphotransfer reaction, with 50% of its phosphoryl group lost within 1 minute of incubation with LytR. LytS has also phosphatase activity. Notably, LytR undergoes phosphorylation by acetyl phosphate at a rate that is 2-fold faster than the phosphorylation by LytS. This observation is significant in lieu of the in vivo observations that regulation of the lrgAB operon is LytR-dependent in the presence of excess glucose in the medium. The latter condition does not lead to perturbation of the cell membrane potential but rather to the accumulation of acetate in the cell. Our, study provides for the first time the molecular basis for regulation of lrgAB in a LytR-dependent manner under conditions that do not involve sensing by LytS.

Numerical Modeling of Fluidic Pumping in Micronetworks of Plants

Plant transport mechanisms are of interest in developing micropump for engineering devices. We present a two-dimensional phloem loading and transport model incorporating protein level mechanics with cellular level fluid mechanics. Governing Navier-Stokes, continuity, and Nernst-Planck equations are numerically solved to determine fluid flow and sugar transport. Phloem loading mechanics for active loading is incorporated through a six-state proton sucrose pump kinetic model. The influence of binding rates constants, concentrations, and membrane electrical potential differences on resulting sucrose transport is studied. Numerical results show that increasing rates of the sucrose transporter will noticeably increase outflow. Simulation result also show that a lower leaf sieve sucrose concentration improves outflow. In addition, a more negative membrane electrical potential difference will increase outflow. This numerical model offers insight on parameters that may be significant for implementing plant transport mechanisms in microfluidic devices.

Study of Protein Facilitated Water and Nutrient Transport in Plant Phloem

Biological systems use transporter proteins to create concentration gradients for a variety of purposes. In plant, sucrose transporter proteins play a vital role in driving fluid flow through the phloem by generating chemical potential. In this study, we investigate these nanoscale phenomena of protein directed active transport in a microscale biological system. We presented a mathematical model for protein facilitated sucrose loading considering six different states of the sucrose transporter protein. In addition, we developed a quasi-one dimensional transport model to study protein facilitated pumping mechanisms in plant phloem. Here we specifically study the influence of transporter protein reaction rates, apoplast proton concentration, membrane electrical potential, and cell membrane hydraulic permeability on flow through the phloem. This study reveals that increasing companion cell side deprotonation rate significantly enhances the sieve tube sugar concentrations, which results in much higher water transport. Lower apoplast pH increases the transport rate, but the flow control is less noticeable for a pH less than 5. A more negative membrane electrical potential difference will significantly accelerate the transporter proteins' ability to pump water and nutrients. Higher companion cell and sieve element membrane hydraulic permeability also promotes flows through the phloem; however, the flow difference is less noticeable at higher permeabilities when near typical plant cell membrane ranges.

Membrane potential differences and viability of grapevine root cells treated with HgCl2

The effect of mercury (Hg) on the electrophysiological and permeability properties of grapevine adventitious root cells was examined. In short-term experiments, the apical segments of adventitious roots were treated with different concentrations of mercury (0.01 µmol/L to 2 µmol/L HgCl₂), and trans-membrane electrical potential differences (E_M) were monitored in root cortical cells, localized in distinct root zones. Based on Hg-induced decrease of E_M, we can confirm that the depolarization of the membrane is an instant Hg-response and the extent of E_M decrease is not only time- and concentration dependent, but also related to the developmental stage of cells and their localization on root axis. The sensitivity of root cells to Hg declined in the direction of cell division zone > cell elongation zone > absorption zone. Long-term treatment of roots with 2 µmol/L mercury showed that Hg-induced decrease of E_M was accompanied by an increase of K⁺ efflux and a decrease of the diffusion potential (E_D) values. Application of fusicoccin (H⁺-ATPase activator) to the root medium caused an immediate hyperpolarization of the membrane in control and Hg-treated cells. Laser scanning confocal microscopy analysis confirmed that Hg reduced cell viability, which was accompanied with the occurrence of cell death hallmarks, like condensation of protoplasts, nuclei fragmentation and deposition of granular material.

In GM-CSF Restrain Mitochondria Pathway Apoptosis NF-kB Is Not the Only Way.

After stimulated by GM-CSF, Akt and IkB kinase (IKK) can be phosphorylated by activated PIK3-kinase. Activated IKK phosphorylate IkB, making NF-kB released and translocate to nucleus. Granulocyte-macrophage colony-stimulating factor (GM-CSF) restrain TF-1 cell mitochondria pathway apoptosis through PI3K pathway, so we asked if NF-κB is the only way in this process. All group were plused TF-1 cells 2.5X105/250μl, group A plused hGM-CSF (100ng/ml ) 50μl; group B plused hGM-CSF (100ng/ml) 50 μl and SN50 (50 μmol/L) 20μl; group C plused hGM-CSF 50μl and SN50 200μl. After cultured for 48 hours, all groups were dyed with Annexin and PI, analyzed in FCM. At the same time all groups were dyed by diluted MitoCapture to analyze mitochondria membrane electrical potential. Drop experimental group B 10μl on slips, so as group C, then observe it under Fluorescence microscopes FL1 channel. Group A cells that have normal mitochondria membrane electrical potential can form poly-MitoCapture in it, and inspired red fluorescence. Group C cells that have monomer MitoCapture was inspired green fluorescence. The red fluorescence intensity of control, group A, B, C means is 368.93, 1137.59, 1033.61 and 618.37 respectively. Group A and B retained a statistically significant difference with control (P<0.01). Group C retained a statistically significant difference with control (P<0.01), group A and group B (P<0.01). This investigation proves that GM-CSF restrain TF-1 cells can be incompletely blocked by SN50, which is the specificity inhibitor of NF-kB. So we get the conclusion that NF-kB is an important but not the only pathway in the process of GM-CSF restrain TF-1 cells apoptosis. It also suggests that there maybe some other pathway in this process.