The developmental and iron nutritional pattern of PIC1 and NiCo does not support their interdependent and exclusive collaboration in chloroplast iron transport in Brassica napus

Abstract

The accumulation of NiCo following the termination of the accumulation of iron in chloroplast suggests that NiCo is not solely involved in iron uptake processes of chloroplasts. Chloroplast iron (Fe) uptake is thought to be operated by a complex containing permease in chloroplast 1 (PIC1) and nickel-cobalt transporter (NiCo) proteins, whereas the role of other Fe homeostasis-related transporters such as multiple antibiotic resistance protein 1 (MAR1) is less characterized. Although pieces of information exist on the regulation of chloroplast Fe uptake, including the effect of plant Fe homeostasis, the whole system has not been revealed in detail yet. Thus, we aimed to follow leaf development-scale changes in the chloroplast Fe uptake components PIC1, NiCo and MAR1 under deficient, optimal and supraoptimal Fe nutrition using Brassica napus as model. Fe deficiency decreased both the photosynthetic activity and the Fe content of plastids. Supraoptimal Fe nutrition caused neither Fe accumulation in chloroplasts nor any toxic effects, thus only fully saturated the need for Fe in the leaves. In parallel with the increasing Fe supply of plants and ageing of the leaves, the expression of BnPIC1 was tendentiously repressed. Though transcript and protein amount of BnNiCo tendentiously increased during leaf development, it was even markedly upregulated in ageing leaves. The relative transcript amount of BnMAR1 increased mainly in ageing leaves facing Fe deficiency. Taken together chloroplast physiology, Fe content and transcript amount data, the exclusive participation of NiCo in the chloroplast Fe uptake is not supported. Saturation of the Fe requirement of chloroplasts seems to be linked to the delay of decomposing the photosynthetic apparatus and keeping chloroplast Fe homeostasis in a rather constant status together with a supressed Fe uptake machinery.

Keywords: Chloroplast; Iron deficiency; Iron homeostasis; Leaf development; Supraoptimal iron nutrition.

Fig. 1

Changes in the Fe content of 4th (a) and 6th (b) leaves during the time of treatment. ∆Fe, Fe deficiency; Ctrl, optimal Fe nutrition (control); + Fe, supraoptimal Fe nutrition. Error bars represent SD values. To compare the differences, one-way ANOVAs were performed with Tukey–Kramer post hoc tests on the treatments (P < 0.05; n = 3 × 3 [biological × technical])

Fig. 2

Changes in the Fe content of chloroplasts isolated from the 4th (a) and 6th leaves (b) during the time of treatment. Treatments as in Fig. 1. Error bars represent SD values. To compare the differences, one-way ANOVAs were performed with Tukey–Kramer post hoc tests on the treatments (P < 0.05; n = 3 × 3 [biological × technical])

Fig. 3

Changes in the Chl a + b concentration of 4th (a) and 6th (b) leaves during the time of treatment. Treatments as in Fig. 1. Error bars represent SD values. To compare the differences, one-way ANOVAs were performed with Tukey–Kramer post hoc tests on the treatments (P < 0.05; n = 3 × 3 [biological × technical])

Fig. 4

Changes in the excitation energy allocation during the time of treatments in the 4th (a) and 6th (b) leaves. Treatments as in Fig. 1. Error bars represent SD values. To compare the differences, one-way ANOVAs were performed with Tukey–Kramer post hoc tests on the treatments (P < 0.05; n = 3 × 3 [biological × technical])

Fig. 5

Correlation between Chl a + b and Fe content of chloroplasts. Linear regression was calculated on 42 averaged individual data pairs where R² is 0.8769

Fig. 6

Correlation between the Fe content of chloroplasts and the actual photochemical quenching of PSII reaction centres (Φ_PSII). Boltzmann fit was calculated on 33 individual averaged data pair where R² was 0.8455

Fig. 7

Changes in the transcript levels of BnPIC1 (a, b), BnNiCo (c, d) and BnMAR1 (e, f). a, c, e 4th leaves. b, d, f 6th leaves. Treatments as in Fig. 1. NRQ, normalized relative quantity of transcript. Error bars represent SE values. To compare the differences, one-way ANOVAs were performed with Tukey–Kramer post hoc tests on the treatments (P < 0.05; n = 3 × 2 [biological × technical])

Fig. 8

Relative amount of NiCo based on immunoblot analysis. a 4th leaves. b 6th leaves. Treatments as in Fig. 1. Error bars represent SD values. To compare the differences, one-way ANOVAs were performed with Tukey–Kramer post hoc tests on the treatments (P < 0.05; n = 3 × 2 [biological × technical])