Transient Influx of nickel in root mitochondria modulates organic acid and reactive oxygen species production in nickel hyperaccumulator Alyssum murale

Abstract

Mitochondria are important targets of metal toxicity and are also vital for maintaining metal homeostasis. Here, we examined the potential role of mitochondria in homeostasis of nickel in the roots of nickel hyperaccumulator plant Alyssum murale. We evaluated the biochemical basis of nickel tolerance by comparing the role of mitochondria in closely related nickel hyperaccumulator A. murale and non-accumulator Alyssum montanum. Evidence is presented for the rapid and transient influx of nickel in root mitochondria of nickel hyperaccumulator A. murale. In an early response to nickel treatment, substantial nickel influx was observed in mitochondria prior to sequestration in vacuoles in the roots of hyperaccumulator A. murale compared with non-accumulator A. montanum. In addition, the mitochondrial Krebs cycle was modulated to increase synthesis of malic acid and citric acid involvement in nickel hyperaccumulation. Furthermore, malic acid, which is reported to form a complex with nickel in hyperaccumulators, was also found to reduce the reactive oxygen species generation induced by nickel. We propose that the interaction of nickel with mitochondria is imperative in the early steps of nickel uptake in nickel hyperaccumulator plants. Initial uptake of nickel in roots results in biochemical responses in the root mitochondria indicating its vital role in homeostasis of nickel ions in hyperaccumulation.

FIGURE 1.

Comparison of tolerance, root absorption, and translocation of nickel in hyperaccumulator A. murale and non-accumulator A. montanum. A, relative root growth increment in Alyssum species in response to various concentrations of nickel in solution culture after 10 days exposure. Values are mean ± S.D. B, percentage inhibition in growth in Alyssum species in response to various concentrations of nickel in solution culture after 10 days exposure. Values are mean ± S.D. Relative nickel accumulations in the roots (C) and shoots (D) of Alyssum species w.r.t treatment duration. Plants of both the species were treated with 700 μm Ni(NO₃)₂ for different time intervals. Roots and shoots of treated and control plants were sampled and nickel was estimated using ICP-AES as described under “Experimental Procedures.” Significant differences among the treatments at p < 0.05 were determined by one-way analysis of variance followed by Tukey-Kramer test (α = 0.5) and are indicated by different letters. Values are mean ± S.D.

FIGURE 2.

Temporal variability in intracellular nickel sequestration in the roots of Alyssum species. Confocal micrographs showing intracellular sequestration of nickel in the roots of A. murale (A) and A. montanum (B) at treatments of 20 min (center panels) and 1 h (bottom panels). Controls (top panels) are representative images for no nickel treatments. The roots were treated with 700 μm Ni(NO₃)₂ and intracellular nickel was visualized with nickel specific dye Newport Green DCF (488 nm) as shown in left panels. Right panels show differential interference contrast (DIC) phase images. Scale bar = 5 μm.

FIGURE 3.

Transient influx of nickel ions in mitochondria and sequestration to vacuoles. Confocal micrographs showing divergence of fluorescence from nickel specific Newport Green DCF and mitochondria-specific MitoTracker Red in the roots of A. montanum (top panel) and A. murale (bottom panel). Dual emission confocal images of Newport Green DCF and MitoTracker Red CMXRos are shown in the left two panels. These images are overlaid in the two rightmost panels to show the deviation in the patterns of the labeled organelles (Overlay). Arrow bars in right panel indicate dual staining of mitochondria in the roots of A. murale. The roots were treated with 700 μm Ni(NO₃)₂ for 1 h before subjecting to staining with dyes. Scale bar = 10 μm.

FIGURE 4.

Confirmation and quantification of mitochondrial nickel influx in purified mitochondria. Confocal micrographs showing co-localization of fluorescence from nickel-specific Newport Green DCF and mitochondria-specific MitoTracker Red CMXRos in purified mitochondrial fractions from the roots of A. murale (A) and A. montanum (B). Dual emission confocal images of Newport Green DCF and MitoTracker Red CMXRos are shown in left two panels. For A. murale, the inset images show co-labeled enlarged mitochondria. These images are overlaid in the right panel to show the coincidence of the labeled organelles (Overlay). Scale bar, 10 μm. C, nickel content in root mitochondria of Alyssum species treated with 700 μm Ni(NO₃)₂ was estimated using ICP-AES. Quantitative data shows increased nickel content in mitochondria of A. murale compared with A. montanum plants. Data are expressed as nanamole of nickel mg⁻¹ mitochondrial protein and represent the mean ± S.D. Data are representative of three experiments, p < 0.05, n = 6.

FIGURE 5.

Comparison of the effects of nickel ions on Krebs cycle in the root mitochondria of Alyssum species. A, a kinetic investigation of citrate synthase (CS, EC 1.11.1.6) in the root mitochondrial fractions of Ni(NO₃)₂-treated (700 μm) hyperaccumulator A. murale and comparison with A. montanum. B, a kinetic investigation of malate dehydrogenase activity in the direction of NADH oxidation (MDH, EC 1.1.1.37) in the root mitochondrial fractions of Ni(NO₃)₂-treated (700 μm) hyperaccumulator A. murale and comparison with A. montanum. C, a kinetic investigation of malate dehydrogenase activity in the direction of malate oxidation (EC 1.1.1.37) in the root mitochondrial fractions of Ni(NO₃)₂-treated (700 μm) hyperaccumulator A. murale and comparison with A. montanum. Total enzymatic activities are expressed as nanamolar/min/mg of mitochondrial protein. Results are the average of three independent determinations ± S.D. *, indicates significant difference from control, p < 0.05).

FIGURE 6.

Malic acid reduces generation of ROS in nickel-treated Alyssum roots. A, intracellular ROS production was monitored with H₂DCFDA in control and treated roots and compared with ROS generated in the roots preincubated with malic acid (MA). Images were acquired using identical parameters on a Zeiss Meta 510 confocal microscope using a ×10 objective; post-processing and analysis was done with ImageJ. Scale bar = 50 μm. B, quantization of fluorescence from images by using ImageJ. RFU, relative fluorescence unit. Significant differences among the treatments at p < 0.05 were determined by one-way analysis of variance followed by Tukey-Kramer test (α = 0.5) and are indicated by different letters.

FIGURE 7.

Schematic of the proposed model for the role of mitochondrial nickel influx in tolerance and hyperaccumulation. The scheme denotes the transient nickel influx in root mitochondria of nickel hyperaccumulator plants, which are proposed to participate in modulation of organic acid and ROS production. The root epithelial cells of nickel hyperaccumulator play a significant role in nickel mobilization and storage in planta. Krebs cycle intermediates, specifically malic acid production, are up-regulated resulting in efflux of malic acid from mitochondria via malic acid shuttle. Intracellular malic acid may bind to nickel ions to form complexes and/or by an unknown mechanism(s) suppresses nickel-induced ROS and prevents apoptosis of the root cells.