Responses of plant calmodulin to endocytosis induced by rare earth elements.

The wide application of rare earth elements (REEs) have led to their diffusion and accumulation in the environment. The activation of endocytosis is the primary response of plant cells to REEs. Calmodulin (CaM), as an important substance in calcium (Ca) signaling systems, regulating almost all of the physiological activities in plants, such as cellular metabolism, cell growth and division. However, the response of CaM to endocytosis activated by REEs remains unknown. By using immunofluorescence labeling and a confocal laser scanning microscope, we found that trivalent lanthanum [La(III)], an REE ion, affected the expression of CaM in endocytosis. Using circular dichroism, X-ray photoelectron spectroscopy and computer simulations, we demonstrated that a low concentration of La(III) could interact with extracellular CaM by electrostatic attraction and was then bound to two Ca-binding sites of CaM, making the molecular structure more compact and orderly, whereas a high concentration of La(III) could be coordinated with cytoplasmic CaM or bound to other Ca-binding sites, making the molecular structure more loose and disorderly. Our results provide a reference for revealing the action mechanisms of REEs in plant cells.

Alterations in cytosol free calcium in horseradish roots simultaneously exposed to lanthanum(III) and acid rain.

The extensive use of rare earth elements (REEs) has increased their environmental levels. REE pollution concomitant with acid rain in many agricultural regions can affect crop growth. Cytosol free calcium ions (Ca(2+)) play an important role in almost all cellular activities. However, no data have been reported regarding the role of cytosol free Ca(2+) in plant roots simultaneously exposed to REE and acid rain. In this study, the effects of exposures to lanthanum(III) and acid rain, independently and in combination, on cytosol free Ca(2+) levels, root activity, metal contents, biomass, cytosol pH and La contents in horseradish roots were investigated. The simultaneous exposures to La(III) and acid rain increased or decreased the cytosol free Ca(2+) levels, depending on the concentration of La(III), and these effects were more evident than independent exposure to La(III) or acid rain. In combined exposures, cytosol free Ca(2+) played an important role in the regulation of root activity, metal contents and biomass. These roles were closely related to La(III) dose, acid rain strength and treatment mode (independent exposure or simultaneous exposure). A low concentration of La(III) (20 mg L(-1)) could alleviate the adverse effects on the roots caused by acid rain, and the combined exposures at higher concentrations of La(III) and acid rain had synergic effects on the roots.

Toxic effects of heavy metal terbium ion on the composition and functions of cell membrane in horseradish roots.

The environmental safety of rare earth elements (REEs), especially the toxic effect of REEs on plants, has attracted increasing attention. However, the cellular mechanism of this toxic effect remains largely unknown. Here, the toxic effects of heavy REE terbium ion [Tb(III)] on the cell membrane of horseradish roots were investigated by using electron microscope autoradiography (EMARG) and histochemical methods. The results indicated that Tb(III) was distributed in the extracellular and intracellular spaces of the roots after horseradish was treated with Tb(III). Moreover, the percentage contents of the unsaturated fatty acids in the membrane lipids, the current of the outward K(+) channel and the average diameter of membrane proteins in the roots of horseradish treated with Tb(III) were decreased; on the contrary, the percentage contents of the saturated fatty acids and malondialdehyde in the roots of horseradish treated with Tb(III) were increased. Furthermore, the contents of intracellular N, P, Mg and Fe in the roots of horseradish treated with Tb(III) were decreased, while the contents of intracellular K and Ca in the roots of horseradish treated with Tb(III) were increased. Finally, the effects of Tb(III) on horseradish roots were increased with increasing concentration or duration of Tb(III) treatment. In conclusion, after horseradish was treated with Tb(III), Tb(III) could enter the cells of horseradish roots and lead to the toxic effects on horseradish, which caused the oxidation of the unsaturated fatty acids in the membrane lipids, the changes in the membrane proteins (including the outward K(+) channel), the decrease in the membrane fluidity, and then the inhibition of the intracellular/extracellular-ion exchange in horseradish roots.

Effects of terbium (III) on signaling molecules in horseradish.

Rare earth elements, especially terbium (Tb), are high-valence heavy metal elements that accumulate in the environment, and they show toxic effects on plants. Signaling molecules regulate many physiological and biochemical processes in plants. How rare earth elements affect signaling molecules remains largely unknown. In the present study, the effects of Tb(3+) on some extracellular and intracellular signaling molecules (gibberellic acid, abscisic acid, auxin, H2O2, and Ca(2+)) in horseradish leaves were investigated by using high-performance liquid chromatography, X-ray energy spectrometry, and transmission electron microscopy, and Tb(3+) was sprayed on the surface of leaves. Tb(3+) treatment decreased the auxin and gibberellic acid contents and increased the abscisic acid content. These changes in the contents of phytohormones (gibberellic acid, abscisic acid, and auxin) triggered excessive production of intracellular H2O2. Consequently, the increase in H2O2 content stimulated the influx of extracellular Ca(2+) and the release of Ca(2+) from Ca(2+) stores, leading to Ca(2+) overload and the resulting inhibition of physiological and biochemical processes. The effects outlined above were more evident with increasing the concentration of Tb(3+) sprayed on horseradish leaves. Our data provide a possible underlying mechanism of Tb(3+) action on plants.

Roles of horseradish peroxidase in response to terbium stress.

The pollution of the environment by rare earth elements (REEs) causes deleterious effects on plants. Peroxidase plays important roles in plant response to various environmental stresses. Here, to further understand the overall roles of peroxidase in response to REE stress, the effects of the REE terbium ion (Tb(3+)) on the peroxidase activity and H2O2 and lignin contents in the leaves and roots of horseradish during different growth stages were simultaneously investigated. The results showed that after 24 and 48 h of Tb(3+) treatment, the peroxidase activity in horseradish leaves decreased, while the H2O2 and lignin contents increased. After a long-term (8 and 16 days) treatment with Tb(3+), these effects were also observed in the roots. The analysis of the changes in peroxidase activity and H2O2 and lignin contents revealed that peroxidase plays important roles in not only reactive oxygen species scavenging but also cell wall lignification in horseradish under Tb(3+) stress. These roles were closely related to the dose of Tb(3+), duration of stress, and growth stages of horseradish.

Direct interaction between terbium ion and peroxidase in horseradish at different pH values.

Rare earth elements (REEs) entering plant cells can directly interact with peroxidase in plants, which is the structural basis for the decrease in the activity of peroxidase. Different cellular compartments have different pH values. However, little information is available regarding the direct interaction between REEs and peroxidase in plants at different pH values. Here, we investigated the charge distribution on the surface of horseradish peroxidase (HRP) molecule as well as the interaction of terbium ion (Tb(3+), one type of REEs) and HRP at different pH values. Using the molecular dynamics simulation, we found that when the pH value was from 4.0 to 8.0, a large amount of negative charges were intensively distributed on the surface of HRP molecule, and thus, we speculated that Tb(3+) with positive charges might directly interact with HRP at pH 4.0-8.0. Subsequently, using ultraviolet-visible spectroscopy, we demonstrated that Tb(3+) could directly interact with HRP in the simulated physiological solution at pH 7.0 and did not interact with HRP in other solutions at pH 5.0, pH 6.0 and pH 8.0. In conclusion, we showed that the direct interaction between Tb(3+) and HRP molecule depended on the pH value of cellular compartments.

Living target of Ce(III) action on horseradish cells: proteins on/in cell membrane.

Positive and negative effects of rare earth elements (REEs) in life have been reported in many papers, but the cellular mechanisms have not been answered, especially the action sites of REEs on plasma membrane are unknown. Proteins on/in the plasma membrane perform main functions of the plasma membrane. Cerium (Ce) is the richest REEs in crust. Thus, the interaction between Ce(III) and the proteins on/in the plasma membrane, the morphology of protoplast, and the contents of nutrient elements in protoplast of horseradish were investigated using the optimized combination of the fluorescence microscopy, fluorescence spectroscopy, circular dichroism, scanning electron microscopy, and X-ray energy dispersive spectroscopy. It was found that Ce(III) at the low concentrations (10, 30 µM) could interact with proteins on/in the plasma membrane of horseradish, leading to the improvement in the structure of membrane proteins and the plasma membrane, which accelerated the intra-/extra-cellular substance exchange and further promoted the development of cells. When horseradish was treated with Ce(III) at the high concentrations (60, 80 µM), Ce(III) also could interact with the proteins on/in the plasma membrane of horseradish, leading to the destruction in the structure of membrane proteins and the plasma membrane. These effects decelerated the intra-/extra-cellular substance exchange and further inhibited the development of cells. Thus, the interaction between Ce(III) and proteins on/in the plasma membrane in plants was an important reason of the positive and negative effects of Ce(III) on plants. The results would provide some references for understanding the cellular effect mechanisms of REEs on plants.

Toxic effect of terbium ion on horseradish cell.

The toxic effect of terbium (III) ion on the horseradish cell was investigated by scanning electron microscopy, gas chromatography, and standard biochemical methods. It was found that the activity of horseradish peroxidase in the horseradish treated with 0.2 mM terbium (III) ion decreased and led to the excessive accumulation of free radicals compared with that in the control horseradish. The excessive free radicals could oxidize unsaturated fatty acids in the horseradish cell and then increase the cell membrane lipid peroxidation of horseradish. The increase in the lipid peroxidation could lead to the destruction of the structure and function of the cell membrane and then damage of the horseradish cell. We propose that this is a possible mechanism for the toxic action of terbium in the biological systems.

Effects of heavy metal terbium on contents of cytosolic nutrient elements in horseradish cell.

The wide application of rare earth elements (REEs) has led to the accumulation of REEs in soil and plant. Thereby, the effect of Tb(3+) on the contents of cytosolic nutrient elements in horseradish was investigated with the synchronous detection technique of scanning electron microscope and energy dispersive X-ray spectrometry. It was found for the first time that the foliar spraying treatment of Tb(3+) destroyed the structure of horseradish mesophyll cells, and then changed the contents of the cytosolic nutrient elements in horseradish, especially Ca. The effect of Tb(3+) was increased with increasing the concentration of Tb(3+). The hydroponical treatment of Tb(3+) could not obviously change the structure of protoplast and the contents of the cytosolic nutrient elements in horseradish leaves. The results indicated that the accumulation of Tb(3+) in soil and plant leaves displayed the different toxic effect on plant leaves.

Toxic effect of heavy metal terbium ion on cell membrane in horseradish.

In order to understand the toxic mechanism of terbium ion (Tb(III)) on plants, the subcellular distribution of Tb(III) in horseradish, the effect of Tb(III) on the composition of the fatty acids in the cell membrane, the peroxidation of membrane lipid, the morphological character of protoplast, the cellular ultrastructure in horseradish were investigated using transmission electron microscopic autoradiography, molecular dynamics simulation, gas chromatography, scanning electron microscopy and transmission electron microscopy. The results show that Tb(III) could not enter the horseradish cell in the presence of 5 mgL(-1) Tb(III) and it was distributed on the cell wall and plasma membrane. The behavior caused the decrease in the contents of unsaturated fatty acids and then the increase in the peroxidation of membrane lipid. Thereby the structure of horseradish cell was damaged. The effects of Tb(III) mentioned above were aggravated in horseradish treated with 60 mgL(-1) Tb(III) because Tb(III) could enter the horseradish cell. It was a possible cytotoxic mechanism of Tb(III) on horseradish.

One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (Armoracia rusticana) treated with Tb(III).

One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (Armoracia rusticana) treated with Tb(III) was investigated using some biophysical and biochemical methods. Firstly, it was found that a large amount of Tb(III) can be distributed on the cell wall, that some Tb(III) can enter into the horseradish cell, indicating that peroxidase was mainly distributed on cell wall, and thus that Tb(III) would interact with horseradish peroxidase (HRP) in the plant. In addition, peroxidase bioactivity was decreased in the presence of Tb(III). Secondly, a new peroxidase-containing Tb(III) complex (Tb-HRP) was obtained from horseradish after treatment with Tb(III); the molecular mass of Tb-HRP is near 44 kDa and the pI is about 8.80. Thirdly, the electrocatalytic activity of Tb-HRP is much lower than that of HRP obtained from horseradish without treatment with Tb(III). The decrease in the activity of Tb-HRP is due to the destruction (unfolding) of the conformation in Tb-HRP. The planarity of the heme active center in the Tb-HRP molecule was increased and the extent of exposure of Fe(III) in heme was decreased, leading to inhibition of the electron transfer. The microstructure change in Tb-HRP might be the result of the inhibition effect of Tb(III) on peroxidase activity in horseradish.

Subcellular location of horseradish peroxidase in horseradish leaves treated with La(III), Ce(III) and Tb(III).

The agricultural application of rare-earth elements (REEs) would promote REEs inevitably to enter in the environment and then to threaten the environmental safety and human health. Therefore, the distribution of the REEs ion, (141)Ce(III) and effects of La(III), Ce(III) and Tb(III) on the distribution of horseradish peroxidase (HRP) in horseradish mesophyll cells were investigated with electron microscopic radioautography and transmission electron microscopic cytochemistry. It was found for the first time that REEs ions can enter into the mesophyll cells, deposit in both extra and intra-cellular. Compared to the normal condition, after the horseradish leaves treated with La(III) or Tb(III), HRP located on the tonoplast is decreased and HRP is mainly located on the cell wall, while HRP is mainly located on the plasma membrane after the horseradish leaves were treated with Ce(III). This also indicated that REEs ions may regulate the plant growth through changing the distribution of enzymes.

Effect of chitosan on peroxidase activity and isoenzyme profile in hairy root cultures of Armoracia lapathifolia.

Hairy root cultures of Armoracia lapathifolia established by infection with Agrobacterium rhizogenes LBA 9402 present a level and isoenzyme pattern of peroxidases (POD) comparable to nontransformed roots. Elicitation with chitosan (10, 50, and 100 mg/L) was used in order to improve POD production. Total POD activity increased about 170% after 48 h of treatment with chitosan 100 mg/L. Elicitation effect on soluble and ionically cell-wall-bound POD fractions of A. lapathifolia hairy roots was analyzed. POD activity of the ionically cell-wall-bound protein fraction increased in the presence of chitosan in a dose-response manner. No effect on soluble POD fractions was observed, but the isoenzyme pattern analyzed by isoelectrofocusing showed an increase of an acidic isoenzyme (pI = 3.4) after the elicitation treatment. The ionically cell-wall-bound protein fraction showed only basic isoenzymes, with an increase of an isoenzyme of pI = 8.7, after the elicitation treatment.