Supraoptimal Iron Nutrition of Brassica napus Plants Suppresses the Iron Uptake of Chloroplasts by Down-Regulating Chloroplast Ferric Chelate Reductase

Abstract

Iron (Fe) is an essential micronutrient for plants. Due to the requirement for Fe of the photosynthetic apparatus, the majority of shoot Fe content is localised in the chloroplasts of mesophyll cells. The reduction-based mechanism has prime importance in the Fe uptake of chloroplasts operated by Ferric Reductase Oxidase 7 (FRO7) in the inner chloroplast envelope membrane. Orthologue of Arabidopsis thaliana FRO7 was identified in the Brassica napus genome. GFP-tagged construct of BnFRO7 showed integration to the chloroplast. The time-scale expression pattern of BnFRO7 was studied under three different conditions: deficient, optimal, and supraoptimal Fe nutrition in both leaves developed before and during the treatments. Although Fe deficiency has not increased BnFRO7 expression, the slight overload in the Fe nutrition of the plants induced significant alterations in both the pattern and extent of its expression leading to the transcript level suppression. The Fe uptake of isolated chloroplasts decreased under both Fe deficiency and supraoptimal Fe nutrition. Since the enzymatic characteristics of the ferric chelate reductase (FCR) activity of purified chloroplast inner envelope membranes showed a significant loss for the substrate affinity with an unchanged saturation rate, protein level regulation mechanisms are suggested to be also involved in the suppression of the reduction-based Fe uptake of chloroplasts together with the saturation of the requirement for Fe.

Keywords: Brassica napus; FCR activity; FRO7; chloroplast; iron allocation; iron nutrition; iron uptake.

FIGURE 1

Heat map of the element accumulation of the 4th and 6th leaves of plants grown on deficient (dFe), optimal (oFe) and supraoptimal (sFe) Fe supply. Heat map plot indicates the concentration of elements as deviance from the total corresponding accumulation in oFe leaves at the 21st day of treatment (zero deviance).

FIGURE 2

Fe content of the 4th leaves of plants grown on deficient (dFe), optimal (oFe), and supraoptimal (sFe) Fe supply, represented by open, light grey and dark grey columns, respectively, on a dry weight (DW) basis of leaves. Error bars represent SD values. To compare the differences, one-way ANOVA was performed with Tukey–Kramer post hoc test on the treatments [P < 0.05; n = 3 × 3 (biological × technical)].

FIGURE 3

Fe content of the 6th leaves of plants grown on deficient (dFe), optimal (oFe), and supraoptimal (sFe) Fe supply, represented by open, light grey and dark grey columns, respectively, on a dry weight (DW) basis of leaves. Error bars represent SD values. To compare the differences, one-way ANOVA was performed with Tukey–Kramer post hoc test on the treatments [P < 0.05; n = 3 × 3 (biological × technical)].

FIGURE 4

Expression of BnFRO7 (bar graph, left scale) and Fe content of chloroplasts (scatter plot in red, right scale) in the 4th leaves of plants grown on deficient (dFe), optimal (oFe) and supraoptimal (sFe) Fe supply, represented by open, light grey and dark grey columns, respectively. Error bars represent SD values. To compare differences between times of measurements, one-way ANOVAs were performed with Tukey–Kramer multiple comparison post hoc test (expression: P < 0.05, n = 12; Fe content: P < 0.05, n = 9), indicated by letters (NRQ: a–e; Fe content: u–w).

FIGURE 5

Expression of BnFRO7 (bar graph, left scale) and Fe content of chloroplasts (scatter plot in red, right scale) in the 6th leaves of plants grown on deficient (dFe), optimal (oFe) and supraoptimal (sFe) Fe supply, represented by open, light grey and dark grey columns, respectively. Error bars represent SD values. To compare differences between times of measurements, one-way ANOVAs were performed with Tukey–Kramer multiple comparison post hoc test (expression: P < 0.05, n = 12; Fe content: P < 0.05, n = 9), indicated by letters (NRQ: a–d; Fe content: u–w).

FIGURE 6

Protein sequence alignment of Arabidopsis thaliana FRO7 on the predicted Brassica napus FRO7. Yellow highlights indicate identical amino acid positions. Asterisks indicate identical amino acids; colons indicate conservation between groups of strongly similar properties; dots indicate conservation between groups of weakly similar properties. Colour coding of the amino acids: red [small + hydrophobic (including F)], blue (acidic), magenta (alkaline + H) and green (hydroxyl + sulfhydryl + amine + G).

FIGURE 7

Localisation of BnFRO7-GFP in the Phaseolus vulgaris chloroplasts detected by confocal microscopy of immunolabelled chloroplasts decorated with anti-GFP antibody. (A–D) Wild type and BnFRO7-GFP containing chloroplasts; chlorophyll autofluorescence. (E–G) High resolution images of BnFRO7-GFP containing chloroplasts. (H) 3D reconstruction. Reconstruction of the same chloroplasts. Scale bars are 10 μm (A–D), 5 μm (E–G), and 2 μm (H). Colours: chlorophyll (red) and anti-rabbit-IgG Atto 550 conjugated antibody (yellow).

FIGURE 8

Saturation of the Fe uptake of chloroplasts isolated from leaves of plants grown on Fe deficient (dFe—triangle), optimal (oFe—square) and supraoptimal Fe supply (sFe—diamond). Error bars represent SD values (n = 12). Curves represent Boltzmann sigmoidal fits on datasets (n = 12).

FIGURE 9

Ferric chelate reductase (FCR) activity of chloroplast inner envelope (cIE) vesicle fractions isolated from leaves of Brassica napus grown under Fe deficient (dFe—triangle), optimal (oFe—square) and supraoptimal Fe supply (sFe—diamond). Boltzmann sigmoidal fit represents the saturation of the FCR activity. Error bars represent SD values.