Three-Dimensional Reconstruction, by TEM Tomography, of the Ultrastructural Modifications Occurring in <i>Cucumis sativus</i> L. Mitochondria under Fe Deficiency

<div><p>Background</p><p>Mitochondria, as recently suggested, might be involved in iron sensing and signalling pathways in plant cells. For a better understanding of the role of these organelles in mediating the Fe deficiency responses in plant cells, it is crucial to provide a full overview of their modifications occurring under Fe-limited conditions. The aim of this work is to characterize the ultrastructural as well as the biochemical changes occurring in leaf mitochondria of cucumber (<i>Cucumis sativus</i> L.) plants grown under Fe deficiency.</p><p>Methodology/Results</p><p>Mitochondrial ultrastructure was investigated by transmission electron microscopy (TEM) and electron tomography techniques, which allowed a three-dimensional (3D) reconstruction of cellular structures. These analyses reveal that mitochondria isolated from cucumber leaves appear in the <i>cristae junction model</i> conformation and that Fe deficiency strongly alters both the number and the volume of cristae. The ultrastructural changes observed in mitochondria isolated from Fe-deficient leaves reflect a metabolic status characterized by a respiratory chain operating at a lower rate (orthodox-like conformation) with respect to mitochondria from control leaves.</p><p>Conclusions</p><p>To our knowledge, this is the first report showing a 3D reconstruction of plant mitochondria. Furthermore, these results suggest that a detailed characterization of the link between changes in the ultrastructure and functionality of mitochondria during different nutritional conditions, can provide a successful approach to understand the role of these organelles in the plant response to Fe deficiency.</p></div

Physiological characterization of leaves from cucumber plants grown in control condition (C) or under Fe deficiency (–Fe).

<p><b>(A)</b> Phenotype of 10 days old cucumber plants grown under control condition (C) or under Fe deficiency (-Fe). The following parameters have been evaluated, on expanded leaves of plants grown as in (A): <b>(B)</b> total chlorophyll concentration, expressed as mg chlorophyll g FW^-1; <b>(C)</b> O₂ evolution, expressed as μmol O₂ min^-1 g FW^-1; <b>(D)</b> net photosynthesis expressed as μmol CO₂ consumed s^-1 m^-2; <b>(E)</b> evapotranspiration, expressed as mmol H₂O s^-1m^-2; <b>(F)</b> stomatal conductance, expressed as mmol CO₂ s^-1m^-2; <b>(G)</b> manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), molybdenum (Mo) concentrations, expressed as μg g DW^-1. Data are means ± SE of at least three independent experiments. Student t-test was used to analyse statistical significance with respect to controls. *:p<0,05; **:p<0,01; ***:p<0,001.</p

Mitochondrial functionality of leaves from cucumber plants grown in control conditions (C) or under Fe deficiency (-Fe).

<p><b>(A)</b><i>In vivo</i> O₂ consumption rates expressed as μmol O₂ consumed min^-1 g FW^-1. The total O₂ consumption rate (IR), as well as the residual O₂ consumption, measured by using KCN (inhibiting cytochrome c oxidase) (IR + KCN) or KCN in combination with SHAM (inhibiting alternative oxidase) (IR + KCN +SHAM) were measured. Values represent mean ±SE of three independent measurements. <b>(B)</b> For each growth condition, the residual O₂ consumption (IR +KCN+SHAM), reported in (A), is shown as % value, with respect to correspondent IR. Mitochondrial respiration is the difference between IR and residual O₂ consumption. Student t-test was used to analyse statistical significance with respect to controls. *:p<0,05; **:p<0,01; ***:p<0,001. <b>(C)</b> Mitochondria (M) and chloroplasts (Cp) were purified from control plants by percoll gradient; the Cp and M fractions at 35/80% percoll and 23/40% percoll layers interfaces, respectively, are indicated with arrows in the gradient tubes. The purified Cp and M fractions were tested by western blot, by using antibodies against two protein markers: Toc33 and porin, specific for Cp, and M, respectively. 10 μg total proteins were loaded in each lane. <b>(D)</b> Western blot analysis of three different mitochondrial Fe-containing proteins (AOX, Rieske, cyt c) on mitochondria purified from leaves of control (C) or Fe-deficient (-Fe) plants. Porin was used as loading control; 10 μg total proteins were loaded in each lane. The results are representative of tree independent experiments.</p

Three-dimensional models of leaf mitochondria from cucumber plants grown in control condition (C) or under Fe deficiency (-Fe).

<p>Different colours were used for the rendering of the different suborganellar structures: magenta for inner membranes (IM), blue for outer membranes (OM), green for cristae and red for cristae junctions. <b>(A-D)</b> Tomographic slices of mitochondria from control (A,B) and Fe-deficient (C,D) plants; the inner membrane (IM), the outer membrane (OM), and the matrix enclosed by the IM are indicated; in particular, the rendering of the mitochondria structures from control (B) and Fe-deficient (D) plants, superimposed on the tomographic slices, is reported. <b>(E,F)</b> Three-dimensional models of the mitochondria from control (E) or Fe-deficient (F) plants. <b>(G,H)</b> Details of mitochondrial cristae junctions, in red, identified in mitochondrial from control (G) or Fe-deficient (H) plants. Scale bars 100 nm.</p

Determination of the cristae number <i>per</i> mitochondrion and the relative intracristae surface area (intracristae surface area <i>per</i> mitochondrion surface area, expressed as % ratio).

<p>Mean values ± SD are from analysis of one hundred mitochondria randomly selected from inclusions obtained from three independent biological samples. Student t-test was used to analyse statistical significance with respect to controls.</p><p>***:p<0,001</p><p>Determination of the cristae number <i>per</i> mitochondrion and the relative intracristae surface area (intracristae surface area <i>per</i> mitochondrion surface area, expressed as % ratio).</p

Transmission electron microscopy (TEM) analysis of leaf tissues from cucumber plants grown in control condition (C) or under Fe deficiency (-Fe).

<p>Control leaf tissues (<b>A-C</b>) and -Fe leaf tissues (<b>D-F</b>). All scale bars = 500 nm. M: mitochondria; C: chloroplast; V: vacuole.</p