Regulation of Iron Homeostasis and Use in Chloroplasts

Abstract

Iron (Fe) is essential for life because of its role in protein cofactors. Photosynthesis, in particular photosynthetic electron transport, has a very high demand for Fe cofactors. Fe is commonly limiting in the environment, and therefore photosynthetic organisms must acclimate to Fe availability and avoid stress associated with Fe deficiency. In plants, adjustment of metabolism, of Fe utilization, and gene expression, is especially important in the chloroplasts during Fe limitation. In this review, we discuss Fe use, Fe transport, and mechanisms of acclimation to Fe limitation in photosynthetic lineages with a focus on the photosynthetic electron transport chain. We compare Fe homeostasis in Cyanobacteria, the evolutionary ancestors of chloroplasts, with Fe homeostasis in green algae and in land plants in order to provide a deeper understanding of how chloroplasts and photosynthesis may cope with Fe limitation.

Keywords: Chlamydomonas; Cyanobacteria; Fe–S; chloroplast; green lineage; iron homeostasis; photosynthesis; plants.

Figure 1

Conceptual model of relationship between atmospheric oxygen and Fe availability during Earth’s history. Percent atmospheric oxygen is presented on the y-axis with time on the x axis. The early earth’s atmosphere contained about 0.001% oxygen until oxygenic photosynthesis began in Cyanobacteria about 2.7 billion years ago (BYA). The increase in oxygen formation led to lowered availability of Fe. Banding iron formations in strata are thought to represent times of intermittent Fe oxide formation from free oceanic Fe as atmospheric oxygen began to increase. Banding iron formations are not found prior to 3 BYA, suggesting the low oxygen content allowed for free Fe. Banding iron formation frequency peaks around 2.5 BYA and these are again not found following 1.8 BYA suggesting that Fe oxidation became constant. The start of soil Fe oxidation is dated to around 2.3 BYA. Timeline depicts billions of years ago. Blue line represents the general trend of increasing oxygen to current levels. Red gradient represents estimated amount of free Fe²⁺ based on frequency of banding Fe patterns in strata with darker red representing more free Fe²⁺. Dotted line represents onset of soil Fe oxidation in the geological record. First occurrence of Cyanobacteria (C), the Great Oxygen Event (GOE) and land plants (LP) are noted along the x axis. Trends are based on data reviewed in [3,9].

Figure 2

Biosynthesis of Fe cofactors in the chloroplast. (a) Tetrapyrrole biosynthesis. Tetrapyrrole biosynthesis produces heme, siroheme, and chlorophyll. Enzymes requiring Fe–S clusters are denoted. Tetrapyrrole biosynthesis begins with Glutamate (Glu) which is used to form aminolevulinic acid (ALA) which is converted into protoporphyrin IX (PPIX). The pathway then splits to either produce heme by insertion of Fe by Ferrochelatase (FeCH) or chlorophyll a and b by insertion of Mg by Magnesium Chlelatase (MgCH). Chlorophyll biosynthesis is catalyzed, in part, by the enzymes, Mg Proto IX Monomethyl Ester Cyclase (MgCy) and Chlorophyllide A Oxygenase (CAO) which require Fe–S clusters. Siroheme cofactor production branches before protoporphyrin IX is produced and is catalyzed by Sirohydrochlorin Ferrochelatase (SirB). Each arrow signifies one enzymatic step. (b) Sulfur Utilization Factor (SUF) Fe–S assembly. Fe–S assembly begins with cysteine desulferase via SUFS and SUFE. The Fe–S cluster is produced on the SUFBCD scaffold and then may be transferred to candidate carrier molecules, including SUFA, High Chlorophyll Fluorescence 101 (HCF101), Nitrogen Fixation U-Like (NFU), and monothiol glutaredoxins (GRX), for delivery to target proteins. Enzymes necessary for cysteine desulfurase are orange, enzymes of the major scaffold are green, and transfer proteins are black. Dashed lines for the carrier proteins indicate biochemical evidence of their role. Solid lines indicate genetic evidence of their role.

Figure 3

Fe-responsive proteins with relevance for photosynthesis in Cyanobacteria, Chlamydomonas, and land plants. Proteins that are discussed in this review for each species are presented (a) Fe-responsive proteins in Cyanobacteria, relatives to the ancestors of chloroplasts. On the outer membrane, Fe³⁺ is chelated by a siderophore and taken up through a TonB Dependent Transporter (TBDT). TonB spans the periplasmic space and the inner membrane to facilitate uptake through the TBDT. On the inner membrane, Fe is taken up as Fe³⁺ via the Fe Uptake Transporter, FutABC, system. Fe can also be taken up as Fe²⁺ after reduction, through Ferrous Iron Transporter B (FeoB). Fe is required for the photosynthetic electron transport chain. During Fe deficiency, Iron Stress Induced protein A (IsiA) can protect Photosystem I (PSI) and Ferredoxin (Fd) can be replaced by the non-Fe-requiring Flavodoxin (Fld) in several species. Two operons exist for Fe–S cluster assembly: Iron Sulfur Cluster (isc) and Sulfur Utilization Factor (suf). Fe is sequestered by ferritin molecules: bacterioferritin and MrgA. (b) Fe-responsive proteins in Chlamydomonas reinhardtii. Chlamydomonas takes up Fe³⁺ by a ferric iron permease yeast homologue (FTR1) after Fe²⁺ is oxidized by Ferric Oxidase 1 (FOX1). Fe is sequestered in the vacuole via Vacuolar Iron Transporter 1 (VIT1) and can be exported from vacuoles via Natural Resistance Associated Macrophage-like Protein 3 (NRAMP3). Chloroplast Fe is sequestered by Ferritin (FER). The ROS-scavenging SuperOxide Dismutases (SOD), FeSOD and MnSOD, are regulated in response to Fe deficiency. (c) Fe-responsive proteins in the leaf mesophyll cell with a focus on chloroplast proteins. From the xylem, Fe can be loaded into the phloem by Oligo Peptide Transporter 3 (OPT3) or to the mesophyll cell. For import into the mesophyll cell, Fe is exported from the xylem by Yellow-Stripe Like 1/3 (YSL1/3) presumably in a Fe³⁺-nicocianamine (NA) complex. Fe³⁺ is reduced at the leaf plasma membrane by Ferric Reductase Oxidase 6 (FRO6) and Fe²⁺ is taken up into the cell. Fe is reduced at the chloroplast envelope by FRO7 and taken up into the stroma by Permease in Chloroplasts 1 (PIC1). ATP Binding Cassette (ABC) proteins, ABCI11, ABCI10, and ABCI12 may also take up Fe into the chloroplast. YSL4/6 is proposed to be a chloroplast Fe exporter. Multiple Antibiotic Resistance1 (MAR1) may transport NA or citrate (CA) into the chloroplast to sequester free Fe. FER is also required to sequester Fe. Fe–S clusters are formed by the SUF pathway in the chloroplast and transfer molecules insert these Fe–S clusters into photosynthetic proteins. Heme and chlorophyll are produced in the tetrapyrrole pathway. Many enzymes for chlorophyll and heme production are Fe-responsive, including Genome Uncoupled 5 (GUN5), Glutamyl-tRNA reductase 1 (HEMA1), and Copper Response Defect 1 (CRD1). Within the chloroplast, during Fe deficiency, ROS-scavenging molecules, Stromal Ascorbate Peroxidase (sAPX) and FeSOD are downregulated in Fe deficiency. Catalase (CAT) is maintained or slightly downregulated during Fe deficiency. Fe is sequestered in the vacuole, where it is imported by VIT1 and exported by NRAMP3/4. YSL4/6 may also be a vacuolar Fe exporter. (d) Fe requirement of photosynthetic electron transport chain proteins. Symbols are explained in the legend.

Figure 4

Role of Ferritin (FER) in preventing Haber–Weiss and Fenton reactions that can perpetuate the production of reactive oxygen species (ROS). FER is expressed under normal conditions to prevent accumulation of free Fe and avoid excessive build up of ROS. If FER cannot capture free Fe, Haber–Weiss reactions, which are catalyzed by free Fe, can lead to the build up of hydroxyl radicals (•OH). These reactions occur in two steps. First, Fe²⁺ and oxygen are produced from Fe³⁺ and superoxide. Second, the Fe²⁺ can react with hydrogen peroxide (H2O2) to produce Fe³⁺ and harmful hydroxyl radicals in a Fenton reaction.

Figure 5

Regulation of Fe deficiency responses in photosynthetic organisms. (a) Regulation of Fe deficiency responses in Cyanobacteria. Fe Uptake Regulator (Fur) is the master regulator of Fe responses and acts as a transcriptional repressor under sufficient Fe. Fur represses the Fe uptake machinery as well as Iron Stress Induced protein A (IsiA). During Fe deficiency Fur is degraded by the protease complex, Filamentous H 1/3 (ftsH1/ftsH3). The expression of the Sulfur Utilization Factor (suf) operon is fine-tuned by the combined action of SufR and the small RNA called Iron Stress Activated RNA 1 (IsaR1). SufR protein, when bound to an Fe–S cluster, represses the suf scaffold to avoid excess Fe–S formation. IsaR1 is induced by Fe deficiency and represses transcripts for the sufBCD scaffold during Fe deficiency together with Cytochrome-b₆f (Cyt-b₆f) complex and Ferredoxin (Fd1). Ferredoxin C2 (FdC2) is hypothesized to regulate IsiA. (b) Regulation of Fe deficiency adjustment in Chlamydomonas. The Fe uptake machinery is regulated transcriptionally by Fe-responsive Elements in the promoters of the genes for the ferric iron permease (FTR1) and Ferric Oxidase 1 (FOX1) and by phosphorylation via the Mitogen Activated Protein Kinase (MAPK) pathway. Light Harvesting Complex A3 (Lhca3) undergoes N-terminal processing (N-TP) during Fe deficiency. Translation of PsaA1 (TAA1) stabilizes PSI under normal Fe conditions but the TAA1 protein is degraded during Fe deficiency, but TAA1 mRNA remains. (c) Regulation of Fe deficiency acclimation in the chloroplast. Fe deficiency responses are regulated by Basic Helix Loop Helix (bHLH) transcription factors, and possibly Ethylene Response Factor (ERF) transcription factors. The bHLH transcription factors, IAA-Leucine Resistant 3 (ILR3) and Popeye (PYE) interact to repress ferritin (FER) and NEET. The ubiquitin ligase, Brutus (BTS) negatively regulates bHLH transcription factors. Iron Dependent Regulatory Sequence (IDRS) elements in the promoter of Ferritin (FER1) mRNA repress FER expression under Fe deficiency. The PAP/SAL signaling pathway may influence responses to Fe deficiency. 3′-phosphoadenosine 5′-phosphate (PAP) is a signaling molecule that can move between cellular compartments and inhibit exonucleases (XRNs). SAL1 enzyme regulates levels of PAP. XRNs can degrade Ethylene Response Factor 1 (ERF1) mRNA resulting in less ERF1 protein accumulation. Targets of ERF1 in Fe deficiency have not presently been identified. During Fe deficiency, the Ironman/Fe Uptake Inducing Peptide (IMA/FEP) signaling peptides are transported in the phloem in order to signal upregulation of Fe uptake in the root.