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Main Conclusion: 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.

Chloroplasts, mitochondria and vacuoles represent characteristic organelles of the plant cell, with a predominant function in cellular metabolism. Chloroplasts are the site of photosynthesis and therefore basic and essential for photoautotrophic growth of plants. Mitochondria produce energy during respiration and vacuoles act as internal waste and storage compartments. Moreover, chloroplasts and mitochondria are sites for the biosynthesis of various compounds of primary and secondary metabolism. For photosynthesis and energy generation, the internal membranes of chloroplasts and mitochondria are equipped with electron transport chains. To perform proper electron transfer and several biosynthetic functions, both organelles contain transition metals and here iron is by far the most abundant. Although iron is thus essential for plant growth and development, it becomes toxic when present in excess and/or in its free, ionic form. The harmful effect of the latter is caused by the generation of oxidative stress. As a consequence, iron transport and homeostasis have to be tightly controlled during plant growth and development. In addition to the corresponding transport and homeostasis proteins, the vacuole plays an important role as an intracellular iron storage and release compartment at certain developmental stages. In this review, we will summarize current knowledge on iron transport and homeostasis in chloroplasts, mitochondria and vacuoles. In addition, we aim to integrate the physiological impact of intracellular iron homeostasis on cellular and developmental processes., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Iron homeostasis is an important process for flower development and plant fertility. The role of plastids in these processes has been shown to be essential. To document the relationships between plastid iron homeostasis and flower biology further, a global study (transcriptome, proteome, metabolome, and hormone analysis) was performed of Arabidopsis flowers from wild-type and triple atfer1-3-4 ferritin mutant plants grown under iron-sufficient or excess conditions. Some major modifications in specific functional categories were consistently observed at these three omic levels, although no significant overlaps of specific transcripts and proteins were detected. These modifications concerned redox reactions and oxidative stress, as well as amino acid and protein catabolism, this latter point being exemplified by an almost 10-fold increase in urea concentration of atfer1-3-4 flowers from plants grown under iron excess conditions. The mutant background caused alterations in Fe-haem redox proteins located in membranes and in hormone-responsive proteins. Specific effects of excess Fe in the mutant included further changes in these categories, supporting the idea that the mutant is facing a more intense Fe/redox stress than the wild type. The mutation and/or excess Fe had a strong impact at the membrane level, as denoted by the changes in the transporter and lipid metabolism categories. In spite of the large number of genes and proteins responsive to hormones found to be regulated in this study, changes in the hormonal balance were restricted to cytokinins, especially in the mutant plants grown under Fe excess conditions.

Iron (Fe) is an essential element for human nutrition. Given that plants represent a major dietary source of Fe worldwide, it is crucial to understand plant Fe homeostasis fully. A major breakthrough in the understanding of Fe sensing and signaling was the identification of several transcription factor cascades regulating Fe homeostasis. However, the mechanisms of activation of these cascades still remain to be elucidated. In this opinion, we focus on the possible roles of mitochondria and chloroplasts as cellular Fe sensing and signaling sites, offering a new perspective on the integrated regulation of Fe homeostasis and its interplay with cellular metabolism., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: High-throughput technologies have opened new avenues to study biological processes and pathways. The interpretation of the immense amount of data sets generated nowadays needs to be facilitated in order to enable biologists to identify complex gene networks and functional pathways. To cope with this task multiple computer-based programs have been developed. GeneTrail is a freely available online tool that screens comparative transcriptomic data for differentially regulated functional categories and biological pathways extracted from common data bases like KEGG, Gene Ontology (GO), TRANSPATH and TRANSFAC. Additionally, GeneTrail offers a feature that allows screening of individually defined biological categories that are relevant for the respective research topic., Results: We have set up GeneTrail for the use of Arabidopsis thaliana. To test the functionality of this tool for plant analysis, we generated transcriptome data of root and leaf responses to Fe deficiency and the Arabidopsis metal homeostasis mutant nas4x-1. We performed Gene Set Enrichment Analysis (GSEA) with eight meaningful pairwise comparisons of transcriptome data sets. We were able to uncover several functional pathways including metal homeostasis that were affected in our experimental situations. Representation of the differentially regulated functional categories in Venn diagrams uncovered regulatory networks at the level of whole functional pathways. Over-Representation Analysis (ORA) of differentially regulated genes identified in pairwise comparisons revealed specific functional plant physiological categories as major targets upon Fe deficiency and in nas4x-1., Conclusion: Here, we obtained supporting evidence, that the nas4x-1 mutant was defective in metal homeostasis. It was confirmed that nas4x-1 showed Fe deficiency in roots and signs of Fe deficiency and Fe sufficiency in leaves. Besides metal homeostasis, biotic stress, root carbohydrate, leaf photosystem and specific cell biological categories were discovered as main targets for regulated changes in response to--Fe and nas4x-1. Among 258 differentially expressed genes in response to--Fe and nas4x-1 five functional categories were enriched covering metal homeostasis, redox regulation, cell division and histone acetylation. We proved that GeneTrail offers a flexible and user-adapted way to identify functional categories in large-scale plant transcriptome data sets. The distinguished feature that allowed analysis of individually assembled functional categories facilitated the study of the Arabidopsis thaliana transcriptome.

The membrane-spanning protein PIC1 (for permease in chloroplasts 1) in Arabidopsis (Arabidopsis thaliana) was previously described to mediate iron transport across the inner envelope membrane of chloroplasts. The albino phenotype of pic1 knockout mutants was reminiscent of iron-deficiency symptoms and characterized by severely impaired plastid development and plant growth. In addition, plants lacking PIC1 showed a striking increase in chloroplast ferritin clusters, which function in protection from oxidative stress by sequestering highly reactive free iron in their spherical protein shell. In contrast, PIC1-overexpressing lines (PIC1ox) in this study rather resembled ferritin loss-of-function plants. PIC1ox plants suffered from oxidative stress and leaf chlorosis, most likely originating from iron overload in chloroplasts. Later during growth, plants were characterized by reduced biomass as well as severely defective flower and seed development. As a result of PIC1 protein increase in the inner envelope membrane of plastids, flower tissue showed elevated levels of iron, while the content of other transition metals (copper, zinc, manganese) remained unchanged. Seeds, however, specifically revealed iron deficiency, suggesting that PIC1 overexpression sequestered iron in flower plastids, thereby becoming unavailable for seed iron loading. In addition, expression of genes associated with metal transport and homeostasis as well as photosynthesis was deregulated in PIC1ox plants. Thus, PIC1 function in plastid iron transport is closely linked to ferritin and plastid iron homeostasis. In consequence, PIC1 is crucial for balancing plant iron metabolism in general, thereby regulating plant growth and in particular fruit development.

In chloroplasts, the transition metals iron and copper play an essential role in photosynthetic electron transport and act as cofactors for superoxide dismutases. Iron is essential for chlorophyll biosynthesis, and ferritin clusters in plastids store iron during germination, development, and iron stress. Thus, plastidic homeostasis of transition metals, in particular of iron, is crucial for chloroplast as well as plant development. However, very little is known about iron uptake by chloroplasts. Arabidopsis thaliana PERMEASE IN CHLOROPLASTS1 (PIC1), identified in a screen for metal transporters in plastids, contains four predicted alpha-helices, is targeted to the inner envelope, and displays homology with cyanobacterial permease-like proteins. Knockout mutants of PIC1 grew only heterotrophically and were characterized by a chlorotic and dwarfish phenotype reminiscent of iron-deficient plants. Ultrastructural analysis of plastids revealed severely impaired chloroplast development and a striking increase in ferritin clusters. Besides upregulation of ferritin, pic1 mutants showed differential regulation of genes and proteins related to iron stress or transport, photosynthesis, and Fe-S cluster biogenesis. Furthermore, PIC1 and its cyanobacterial homolog mediated iron accumulation in an iron uptake-defective yeast mutant. These observations suggest that PIC1 functions in iron transport across the inner envelope of chloroplasts and hence in cellular metal homeostasis.