UCLA

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.