In vertebrates, hepcidin is the peptide hormone that homeostatically regulates the body's supply of elemental iron, an indispensible reactant in many biological processes. Worldwide over a billion people are affected by diseases associated with iron imbalance. These conditions can be categorized as disorders in which hepcidin responds physiologically and disorders in which hepcidin is dysregulated. In the former, lack of dietary iron appropriately suppresses hepcidin to maximize dietary iron absorption capacity. Iron-restricted anemia can develop, nevertheless, if the iron content of food is not sufficient. In the latter, inappropriate hepcidin stimulation by cytokines, during chronic inflammation for example, reduces plasma iron and anemia results despite adequate levels of dietary and body iron.

Iron absorption depends on the interaction between hepcidin and its receptor, the iron exporting channel, ferroportin. When hepcidin activity is insufficient, iron pathways saturate and excess iron intoxicates the organs in which it accumulates. When plasma iron levels need to be reduced, hepcidin is secreted by the liver into circulation where it finds ferroportin and initiates its internalization and degradation. Plasma iron flow is thus blocked from the principle ferroportin-expressing cell types including, but not limited to, enterocytes, macrophages of the spleen and liver and hepatocytes until an unknown signaling mechanism suppresses hepcidin production to allow iron to flow to sites of need.

How the liver moderates hepcidin production in response to increased body iron is still not completely understood, but Chapter 1 of this dissertation, describes the use of mouse models of hepcidin deficiency to define two signals that independently regulate hepcidin production. Chapter 2 builds on a collaboration described in Chapter 3, and details the optimization and use of hepcidin mimetics to treat iron overload caused by hepcidin deficiency. Chapter 4 is another collaboration that elucidates novel signaling pathways that suppress hepcidin and their connection to iron overload in conditions that damage cells of the liver.

ABSTRACT

One-sentence summary

The ability to adapt to a changing climate while sustaining the nutritional needs of a growing human population are major challenges of modern human history. Basic plant research and plant biotechnology are key solutions to meeting both challenges. However, iron (Fe)-deficiency both in the oceans and on arable land undermines those efforts. Iron is an essential nutrient for growth and energy production in all forms of life as a cofactor in a myriad of enzymatic reactions. When environmental Fe availability can no longer support intracellular Fe demands, photosynthetic organisms degrade Fe-dependent proteins, especially proteins involved in photosynthesis, to reduce the intracellular Fe content. Photosystem I (PSI) is a primary target for this degradation owing to its high Fe demands within three Fe4S4 clusters. Therefore, the mechanism by which photosynthesis is maintained with limited photosynthetic components remains an area of great research interest. Nevertheless, certain essential Fe-dependent processes are maintained for “housekeeping functions” to allow for cell maintenance. The metabolic pathway that is preserved or degraded in Fe limitation suggests a hierarchy of Fe-dependent processes which I systematically investigated in the following chapters of this dissertation. To better understand the hierarchy of Fe-dependent processes in the face of Fe-limitation, I examined the transcriptome, proteome, protein structure, and physiology of a well-studied, freshwater alga, Chlamydomonas reinhardtii (Chlamydomonas), and two extremophile, marine algae, Dunaliella salina Bardawil and Dunaliella tertiolecta, as they transitioned into Fe-starvation. I studied this with three specific aims: 1) Capturing the temporal view of Chlamydomonas acclimating from an Fe-replete condition to Fe limitation using transcriptomics; 2) Cataloging Fe-binding proteins from Chlamydomonas, D. salina Bardawil, and D. tertiolecta and exploring their responses to both trophic and Fe nutrition stages through proteomics; and 3) Discovering an Fe deficiency induced structural modification to PSI in D. salina Bardawil and D. tertiolecta. In Aim 1, Chlamydomonas was transferred from an Fe-replete to an Fe-limited medium where samples were taken for RNA-seq analysis to capture both short- and long-term transcriptome changes over 48 h. The analysis revealed a hierarchy among the pathways responding to Fe starvation. Among the Fe transporters, FRE1 transcripts were the most highly upregulated (10,000-fold increase) within 30 min in Fe-starved medium, and this was a result of transcriptional regulation shown with a promoter-reporter analysis. Of the bioenergetic pathways, transcripts for chlorophyll biosynthesis and photosynthetic components were the first to respond to low Fe medium while transcripts for respiratory components responded last and were comparatively maintained. I supplemented the transcriptomic observations with measurements for protein abundance and physiology such as chlorophyll content, intracellular metal content, and growth. The physiology measurements showed Chlamydomonas experienced the distinct Fe nutrition stages of Fe-replete, Fe deficiency, and Fe limitation over the 48 h experiment. In Aim 2, I cataloged Fe-binding proteins from Chlamydomonas, D. salina Bardawil, and D. tertiolecta using both computational predictions and orthology to known Fe-binding proteins with manual curation. Proteomics of Chlamydomonas grown in two different trophic conditions (mixotrophic and phototrophic) and four different Fe nutritional stages (Fe-limited, Fe-deficient, Fe-replete, and Fe-excess) were used to probe the different roles of Fe-containing proteins in the cell. D. salina Bardawil and D. tertiolecta Fe-binding proteins were also investigated using proteomics from cells grown in Fe-replete and Fe-starved conditions. Through this analysis, we identified 388, 292, and 277 Fe-containing proteins in C. reinhardtii, D. salina Bardawil, and D. tertiolecta, respectively. Finally, in Aim 3, I used single-particle cryogenic electron microscopy to unveil the Fe-starved PSI structure from D. salina Bardawil and D. tertiolecta. This Fe-starved PSI-light harvesting complex (LHC) I supercomplexes included a second LHCI belt with a novel chlorophyll-binding protein called TIDI1 (thylakoid iron deficiency induced protein) compared to the Fe-replete PSI-LHCI supercomplexes. Altogether, my studies provide a comparative view of both dynamic and static acclimation strategies to low Fe nutrition in both a well-studied alga, Chlamydomonas, and two extremophile, marine algae, D. salina Bardawil and D. tertiolecta, and these chapters showcase the diversity of the responses to Fe limitation among green algae.

Iron (Fe) is vital to growth and energy production of living organisms because it serves as a critical cofactor in many enzymatic reactions. Understanding Fe homeostasis in the plant lineage, including Fe storage and recycling mechanisms, provides a pathway to improve global primary productivity in agriculture and carbon capture capabilities. Chlamydomonas reinhardtii is a eukaryotic, unicellular green alga that has been used as a photosynthetic reference organism for the study of Fe metabolism, biofuel production, and other processes. In many organisms, Fe storage is often realized using vacuoles or the prototypical Fe storage protein, ferritin. In Chlamydomonas, ferritin appears to play a minor role in Fe storage, as its abundance is reduced with increasing amounts of extracellular Fe. Separately, the acidocalcisome, a lysosome-related vacuole characterized by acidic pH, high calcium (Ca) and polyphosphate (polyP) content, has been shown to house various trace metal ions in over-accumulating situations and is a candidate reservoir for Fe storage. Yet, the exact location for excess Fe in Chlamydomonas is uncertain. The pathway for and the regulation of sequestration, distribution, and mobilization of excess Fe taken up under Fe luxury conditions are not well understood either. Targeting these issues, my project was set to address three specific aims on Fe storage and transport in Chlamydomonas: 1) Systematic analysis of secondary experimental variables that determine cellular Fe accumulation; 2) Distinguishing the role of acidocalcisomes in Fe storage; and 3) Identifying the molecular mechanisms for excess Fe sequestration and mobilization. In Aim 1, six common experimental variables relating to various environmental aspects were surveyed to examine their impact on cellular growth and Fe content. The results revealed that cells over-accumulate Fe during stationary phase (3-fold increase) and in alkaline condition (10-fold increase). These Fe over-accumulating conditions were used to address questions in Aim 2 and 3. In Aim 2, various types of elemental images consistently showed Fe in cells grown at alkaline pH colocalizing strongly with Ca and P, markers of acidocalcisomes. In contrast, the Fe accumulated in cells during stationary phase is mostly sequestered into foci that do not contain Ca or P, suggesting an Fe storage site other than the acidocalcisome. Thus, cells may be selectively housing Fe in the acidocalcisome under specific conditions. Interestingly, investigation over the bioavailability of stored Fe suggested that the Fe accumulated under alkaline condition is not readily accessible, as indicated by the slow growth of cells from such cultures upon transfer to Fe-free medium. In Aim 3, a comparative transcriptomic analysis of cells grown in alkaline vs. neutral media exposed a significant increase (138-fold) in the abundance of FEA2 transcripts, encoding a periplasmic Fe-assimilating protein, hence strongly implicating FEA2 in playing a specific role in Fe accumulation under alkaline condition. In addition, a set of ten mutants involved in Fe homeostasis was tested for the capability to accumulate and re-distribute excess Fe. Surprisingly, the fer1 strain, lacking the predominant ferritin subunit, over-accumulated even more Fe than did the wild-type and other tested mutant strains, but failed to benefit from its greater Fe content. This outcome implies that ferritin has a role in Fe acquisition and mobilization. Altogether, my studies provide a methodical analysis of the experimental stimuli resulting in Fe accumulation and the underlying mechanisms of Fe storage and transport in Chlamydomonas at a fundamental level.